JP2020530556A - Sequential lateral flow device - Google Patents

Abstract

The present disclosure provides methods and lateral flow devices for detecting multiple target analytes in a liquid sample. In some practices, the lateral flow device disclosed above includes a housing unit, a capillary flow bed, a sample receiving zone, a buffer receiving zone, and a capture zone. The device is configured to control the flow of the sample and reagent buffer in a sequential fashion with minimal mixing. In some practices, the methods disclosed above can detect multiple target analytes in an assay by applying the binder and signal generator in separate or sequential steps.

Description

Related Applications This application claims the interests of US Provisional Application No. 62 / 537,701 (July 27, 2017), the entire teaching of which is incorporated herein by reference.

Background Lateral flow immunoassay (LFI) is a method for the rapid detection of chemical and biochemical markers for toxicity and disease. LFIs can be divided into three types: (1) competing assays (eg, those used to detect small molecule drugs of abused and therapeutic drugs); (2) proteins, glycoproteins, and lipopoly. Immunometric assays for large molecules such as saccharides (also referred to as "sandwich"assays); and (3) serologic assays that detect antibodies to specific antigens in biological samples. The present disclosure focuses on immunometric assays, in which the term "sandwich" assay is used interchangeably for reference.

Sandwich LFIs (or immunometric LFIs) generally include a highly absorbable flow path on which a capture antibody and a mobilizable detection antibody attached to a signaling agent are immobilized. The signal-generating agent is typically selected from detectable colored and / or fluorescent particles, dyes, and / or enzymes. The term "direct assay" is used when the signaling agent is directly conjugated to the detection antibody.

Several patents and applications describe so-called sequential LFIs. In these applications, multiport devices are typically described as required to accommodate special reagents. In several patents and applications (eg, US Pat. No. 4,981,786, EP 1 044 372 B1, US 2013/0164193), the main use for additional ports is to dye the enzyme attached to the detection antibody. Substrate and / or fluorescence-producing substrate reagents are introduced separately and sequentially. Other applications describe the use of a second port to allow the introduction of wash buffer (eg, US Pat. No. 4,981,786). Although these assays are sequential in the order of manipulation, they are still direct assays in that the signaling agent (eg, enzyme) is directly conjugated to the detection antibody.

In examining the history of LFI from its early concepts in the 1980s to the present day, the use of enzymes as signal-producing moieties on detection antibodies has diminished and is taken by detection antibodies directly bound to signal-producing particles. Has been replaced. Colloidal gold and colored and / or fluorescent latex have been found to be widespread and easy to use. Substrate addition is avoided when such signal-generating particles directly conjugated to the detection antibody are used.

In the process of developing and optimizing the multi-analyte immunometric LFI, we signal generation when more target assays are present for detection through measurements on one-step test strips. We have found that immunometric assays that use detection antibodies that are directly bound to particles have the drawback of reduced sensitivity. For example, if the sandwich LFI test strip detects three different target analytes, a detection limit (LoD) specific to each analyte is obtained. However, its LoD is reduced if three additional target analytes are present in the sample mix (a total of six target analytes). Given that there are three other target analytes in addition to the six target analytes (a total of nine target analytes for detection), the LoD of each target analyte is further exacerbated and ultimately Deteriorates to the point that it becomes less useful.
These limits are in part due to the surface area limits of the signal-producing particles and the limited binding capacity on the particle surface. These and other features of current methods and devices limit their application. The present disclosure provides, among other things, improved methods and devices that address the limitations of current approaches.

U.S. Pat. No. 4,981,786 European Patent No. 1044372 U.S. Patent Application Publication No. 2013/01/64193

Abstract In one aspect, the present disclosure is an improved method for detecting multiple targeted analytes (eg, bacterial, viral, and / or fungal antigens) in a rapid, simple, sensitive, and multi-analytical fashion. And provide devices. The disclosed methods and / or devices are conventional lateral flow devices and / or methods by allowing a binding assay to be performed using well-controlled sequential steps and innovative device features. Overcome some of the challenges and limitations of. In addition, the methods and devices of the present disclosure are used in one assay that uses multiple binders (eg, one or more types or idiotypes of antibodies to many target binding sites (eg, polyclonal antibodies)). It is particularly well suited for the detection of numerous targeted assays (eg, bacterial, fungal, and / or viral antigens). Assay sensitivity is particularly critical in such situations where a large number of antigens have been detected in a complex sample using a polyclonal antibody as the binder.

In one aspect, the present disclosure uses a multi-assay detection method, eg, a sandwich LFI test strip according to some embodiments and / or a lateral flow device according to some embodiments, in a liquid sample of 30, 40 species. , Or an assay capable of detecting as many target analytes as possible. For example, the disclosure is to detect a large number of species and strains of a targeted assay, such as bacterial antigens that can potentially infect platelet preparations intended for therapeutic and / or defensive infusion to a patient. The multi-analyte detection assay of. The present disclosure also provides a rapid assay and / or test (eg, LFI (eg, sandwich LFI)), which is described above, while the culture test requires the start of the test 1-3 days prior to infusion. Since the disclosed assay or test can be performed immediately prior to infusion, it is considered an improvement as a means of safety beyond established culture-based tests.

A single LFI test strip used in conventional methods and / or devices has its various contaminants in clinically relevant LoDs due to restrictions on particle conjugates (or detection antibody-signal-producing particle conjugates). Its ability to detect bacterial species and strains, especially the ability to detect multi-analyte (especially 40 or more targeted analysts), presents great challenges.

In one aspect, the binder (eg, multiple antibodies (eg, one or more types of polyclonal antibodies) are separated from the signal generator (eg, signal generating particles) at the beginning of the LFI test procedure. The binding on the surface of the particles to be detected, however, by allowing the plurality of target assays in the liquid sample to first form a sandwich complex in the capture zone prior to the release of the signal generator. The sensitivity limitation imposed by the limitation of the number of agents is significantly overcome. In this sequential mode, LoD is doubled in the multiplexing assay compared to a similar multiplexing non-sequential direct assay. Can be improved to about 2 log multiples.

In one approach, the formation of a sandwich complex (eg, a complex between the binder and the target analyte, the immobilized capture agent) in the capture zone is that the binder or analyte signals. As long as it happens before contact with the productant. In some practices, the liquid sample is optionally mixed with the detection antibody before being introduced into the lateral flow device. This procedure allows some of the detected antibodies to bind to some of the targeted analytes before entering the flow path. In addition, the number of signal-producing particles required can be reduced to only sufficient to label the actual sandwich complex. As the target analyte is captured by the scavenger immobilized on the flow path, the efficiency of labeling the target analyte with the detection antibody is such that the reaction between the target analyte and the detection antibody is no longer present. It is greatly increased because it is not limited to diffusion. Excess of the detected antibody can flow off the captured target analyte on the flow path for binding. The sensitivity of the assay is therefore not with the binder-signal generator conjugate or the number of signal generators bound to and captured in the capture zone with the binder (eg, detection antibody). It is driven by the number of effective binding events with the captured target assay. Since the binder is much smaller in size than the binder-signal generator conjugate or the conjugate bound to the analyte, higher local concentrations of the binder can be achieved in a given treated sample volume. In some cases, this is an excess of log magnification compared to the maximum number of signal-generating particles that the strip typically contains. In addition, in the non-sequential assay, a large percentage of the binder-signal generator conjugate is captured in the capture zone without the event of binding to the target analyte, causing interference and reducing detection sensitivity. ..

In some practices, the binder and the signal generator in excess were controlled by separating the binder (eg, detection antibody) from the signal generator (eg, signal generating particles). It can be applied or flowed sequentially across the capture zones in a fashion. In this way, a significant improvement in sensitivity can be achieved, for example, using a single LFI strip at the desired LoD, allowing greater diversity of target analytes to be detected. Thus, multiple sandwich complexes can be formed with increasing local concentration of binder, which is then "labeled" with a signal generator.

In addition, the disclosed methods and devices exhibit bacterial contamination in a time-bound manner, eg, in less than a few hours (eg, less than 4 hours, less than 3 hours, less than 2 hours, and / or less than 1 hour). It can be tested to detect multiple target analytes at clinically significant levels with improved sensitivity. This facilitates the testing of blood and / or blood products prior to infusion, for example, without the need for accelerated growth of bacteria and / or viruses in nutrient media, which can take from one to several days. To.

In some aspects, the present disclosure presents an assay in a sequential multi-step fashion (eg, an immunoassay with sequential steps) for the detection / determination of multiple target analytes in a liquid sample (eg, blood or blood product). ) Provide methods and devices for doing so. In an exemplary practice, the disclosed methods detect multi-analytes using binders that are themselves complex (eg, one or more types of polyclonal antibodies).

In one aspect, the present disclosure provides a method for detecting multiple target analytes in a liquid sample using a lateral flow device. The method is under conditions that allow the formation of at least one sandwich complex of the liquid sample and a plurality of scavengers and a plurality of binders placed on a solid support in the lateral flow device. In the first contacting step, the at least one sandwich complex is one or more of the plurality of binders, one or more of the target analytes, and the capture agent. Includes steps involving one or more of them. Each of the above binders is tagged with one member of the conjugate pair. In some practices, the plurality of binders and the plurality of capture agents are antibodies. In some practices, the plurality of binders and the plurality of capture agents are monoclonal antibodies, polyclonal antibodies, and / or mixtures thereof. In some practices, the plurality of binders and the plurality of capture agents are polyclonal antibodies. In some practices, the plurality of binders and / or the plurality of scavengers may bind to at least one antigen that is common to at least a subset of the target analyte. In some practices, the plurality of target analytes may comprise a multipart set or type of target analyte (eg, bacteria (eg, Gram-positive and / or Gram-negative bacteria), viruses, and / or fungi). .. Each type or subset of the target analyte may further include subtypes or different genera. Within each subset or type (or subtype or genus) of the target analyte, there may be a common antigen to which the binder or scavenger can bind. For example, the plurality of targeted analytes may include at least a subset of Gram-positive bacteria and / or a subset of Gram-negative bacteria, and / or subtypes thereof. Each of the binders and / or scavengers may bind at least a partial set of Gram-positive bacteria or an antigen common to a subtype thereof, and / or at least a partial set of Gram-negative bacteria or a subtype thereof. Can bind to antigens common to. Due to the diversity of antigenicity among species, many types of binders can be used. After the formation of the sandwich complex, the signal generator (eg, non-enzymatic signal generator) is such that the signal generator binds to the binder of the sandwich complex (eg, the interaction of the conjugate pair). Under conditions that allow the formation of the detection complex (through), it is introduced into the device and contacted with the sandwich complex. The formation of the detection complex indicates the presence of one or more of the plurality of target analytes. This is because the signal generator produces a detectable signal where the detection complex is formed in the device. The lateral flow device is adapted to inhibit the signal generating agent from contacting the plurality of binders prior to forming the sandwich complex. The signal generated by the formation of the detection complex is detected to determine the presence of the target analyte. In one aspect, the signal generating agent is a non-enzymatic agent, which allows direct detection of the signal without resorting to amplification techniques (eg, PCR amplification or amplification based on the product of the catalytic enzyme reaction). .. In this way, the disclosed methods or devices include a simplified detection step that reduces operating and / or manufacturing costs. Earlier LFIs may have incorporated some sequential treatment of reagents, but this sequential treatment was enforced by the use of enzymatic assays to generate signals in situ (eg, enzymatic signals). Producer). In addition, the enzyme assay can be influenced by factors such as temperature and enzyme / substrate concentration and can be interfered with by the presence of any inhibitor or activator that is not directly related to the determination of the target analyte. Therefore, the step of directly detecting the sandwich complex using a signal generating agent (eg, signal generating particles) can skillfully avoid these potential challenges and may result in false positive results in the detection step or various. Potential interference factors can be reduced.

In some practices, the lateral flow device is a capillary in the solid support that facilitates the flow of the liquid sample and the sample receiving pad in the sample receiving zone for introducing the liquid sample into the lateral flow device. Includes a virtually opaque backing placed between the flow beds. Thus, the backflow of the liquid sample in the proximal direction of the lateral flow device is reduced or minimized. In some embodiments, the lateral flow device comprises a housing unit, wherein the upper inner surface of the housing unit includes a series of ribs to contain liquid beyond the capacity of the sample receiving pad. , Thereby preventing the excess fluid from overflowing the sample receiving pad.

In some practices, the sandwich complex may be formed in one step by flowing a mixture of the liquid sample and the plurality of binders through the capture zone and contacting the capture agent. Under suitable conditions, the sandwich complex is one or more of the plurality of binders, one or more of the target analytes, and one or more of the scavengers. Can be formed with. According to some practices, the sequential flow of samples and reagents through the capture zone to the distal end of the assay strip is the liquid sample and multiple binders (eg, detection antibodies) (which it is. By a separate release and flow of the signal generating agent (eg, signal generating particles) that may adhere to the binder of the sandwich complex in subsequent steps, first flowing (not bound to the signal generating agent). Can be achieved. By doing so, there is little or no mixing of the two sequential fluid flows to help achieve improved sensitivity.

In some practices, the formation of the sandwich complex can be achieved in two steps. The first step involves combining the liquid sample with the plurality of binders in order to form a first complex between at least some of the target analytes and at least some of the binders. Includes contacting steps. In the second step, in order to form the sandwich complex, the first complex and a plurality of scavengers immobilized on the solid support are combined, and the plurality of scavengers are used as the first composite. Includes the step of contacting under conditions that allow it to bind to the body. In some practices, the first complex is simply by a person mixing the liquid sample and a solution containing the plurality of binders in a test tube; and / or the liquid sample, the plurality. Among the devices described herein, such as a lateral flow device preloaded with a binder and / or a device in which the liquid sample is mixed with the plurality of binders inside the device (eg, a device described herein). Can be formed by adding to any of).

In some practices, the step of mixing the liquid sample with the plurality of binders can be performed in a buffer solution at room temperature and / or with slight heating and / or stirring. Sufficient time should be given so that the binding between the target analyte and the binder can be as complete as possible. In some practices, the plurality of binders are 3 or more target analytes (eg, 5, 10, 20, 30, and 40, or more target analytes). ) Can be selected to detect. The disclosed method can detect a larger number of target analyzers by using a sequential flow mechanism without compromising the sensitivity of the test.

In some practices, after the formation of the first complex, the sandwich complex is by flowing a buffer solution containing the first complex over the immobilized scavenger on the solid support. , Can be formed. In one embodiment, the solid support is a lateral flow bed in a lateral flow device (eg, a device described herein). The excess amount of the first complex that does not form the sandwich complex with the scavenger can be removed, for example, by washing the solid support with a buffer solution. Alternatively, the unbound first complex can flow past the location of the immobilized scavenger towards the reservoir in a lateral flow device (eg, a device described herein). Appropriate conditions can be applied, for example, to promote the formation of the sandwich complex by slightly heating the solid support using a buffer solution or adjusting the appropriate pH level.

The formation of the sandwich complex also involves contacting the liquid sample with the plurality of immobilized capture agents to include at least some of the target analytes and at least some of the capture agents. This can be achieved by first forming the body and then contacting the complex with the plurality of binders. In some practices, all of the liquid samples containing the plurality of target analytes can be contacted with the scavenger. Thus, the proximity of the target analyte and the scavenger effectively increases both local concentrations due to the interaction.

Each of the plurality of binders (eg, detection antibody) can be tagged with a small molecule (eg, a small molecule less than about 1000 MW) that is one member of the conjugate pair and the signal generator (eg, eg). The signal-generating particle) can be tagged with the other member of the conjugate pair. For example, biotin can be covalently attached to the binder (eg, detection antibody) and avidin analogs (eg, streptavidin, neutravidin, anti-biotin antibody, etc.) are coated on the surface of the signal-producing particles. Get (or vice versa). In this way, the signal-producing particles can adhere to the detection antibody only after the sandwich complex has been formed, for example, in the capture zone of the test strip. In some practices, the plurality of binders (eg, biotin-labeled detection antibody) are about 1/10 the size of the about 100 nm particle-detection antibody conjugate.

The binder may be one or more types of binder (eg, 2 types, 3 types, 4 types, 5 types, 10 types, 15 types of binders, depending on the number and type of target analytes. , 20, 30, 40, or many different types). In some practices, the binding agent is an antibody, eg, a monoclonal antibody and / or a polyclonal antibody) (eg, at least a partial set of the target analyte (eg, a plurality of Gram-negative and / or in the liquid sample). Contains antibodies that specifically bind to a common antigen of Gram-positive bacteria). The target analyte may include multiple types or subsets of the analyte, within each type or subset a number of subtypes. Each of the above types or subsets or subtypes thereof may have an antigen common to the analytes within the type or subset or subtypes thereof to which the binding agent can specifically bind. The binding agent can be a polyclonal antibody (eg, one or more types of polyclonal antibody). In some practices, the binding agent is at least a portion of Gram-positive and / or Gram-negative bacteria. It specifically binds to at least one antigen common to the set or the subtypes mentioned above. In some practices, the binding agent is more than one or more of polyclonal and monoclonal antibodies. Selected from the type. Each of the binding agents can be associated with the first member of a conjugate pair (eg, a biotin and biotin binding protein pair). The biobinding protein is an avidin, neutral avidin, anti-biotin antibody. , Streptavidin, and other biotin binding proteins.

In some practices, the capture agent comprises one or more types of capture agent (eg, one or more types of polyclonal antibody). For example, the capture agent may be 2 types, 3 types, 4 types, 5 types, 10 types, 15 types, 20 types, 30 types, 40 types, or more of the capture agents, depending on the number and type of the target analyte. Can include many types. In some practices, the scavenger is adapted to specifically bind at least one antigen common to at least a partial set or subtype thereof of the target analyte in the liquid sample. In some practices, the plurality of capture agents are selected from one or more types of polyclonal antibodies and / or monoclonal antibodies. In some practices, the plurality of capture agents comprises one or more capture taxa on the solid support, the taxa being spatially separated from each other and each taxon. Includes at least one scavenger that specifically binds to a variety of common antigens in at least a partial set of target analysts (eg, one antibody that specifically binds to different target analysts). In some practices, at least some of the binders are antibodies of a different type than at least some of the scavengers. In some practices, at least some of the binders are antibodies of the same type as at least some of the scavengers.

The disclosed methods also include the step of forming a detection complex to provide a readable signal to indicate the presence of multiple target analytes in the liquid sample. The formation of the detection complex is accomplished by contacting the sandwich complex with the signal generator under conditions that allow the signal generator to bind to the binder of the sandwich complex. This indicates the presence of one or more of the plurality of target analytes. The signal generated by the signal generator in forming the detection complex is used to determine the presence of the plurality of target analytes. The signal generator is tagged with a second member of the conjugate pair (eg, biotin and biotin-binding protein pair). In some practices, the conjugate pair comprises biotin and a biotin-binding protein, and either the binder or the signal-generating agent is labeled with biotin. In some practices, the biotin-binding protein is selected from the group consisting of avidin, neutravidin, anti-biotin antibody, streptavidin, and other biotin-binding proteins. In some practices, each of the multiple binders is associated with avidin and / or streptavidin. In some practices, the signal generator is selected from the group consisting of metal particles, fluorescent dyes, and latex particles. Binding of the signal-generating agent to the binder of the sandwich complex indicates the presence of the plurality of target analytes in the liquid sample.

By introducing the signal generator after the formation of the sandwich complex, the sensitivity of the method is such that the increasing number of sandwich complexes increases the binding between the binder and the target analyte. Increased if formed prior to signal detection as a result of the event. As used in conventional methods, the limitations imposed by the large size of the binder-signal generator conjugate and the limited binding density have been removed, thereby removing the target analyte. More binds to the binder, allowing the potential to produce more of the sandwich complex for detection. The signal generated by the formation of the detection complex is detected to determine the presence of the plurality of target analytes. Depending on the nature of the signal generator used in the method, the signal generator can be used by a detectable signal (eg, a visual signal, a chemically detectable signal, an electrical signal, and / or an instrument). / Or a signal detectable by a person reporting the formation of a third complex) can be generated. In some practices, the detection is by measuring the optical signal either by the naked eye and / or by an optical instrument. In some practices, the detection is through measuring electrical signals using electrodes in buffer solution in the presence of the third complex. In some practices, the steps of the methods described herein are automated, eg, when a device (eg, a device described herein) is used, and each step can be automated. A person applies the liquid sample to the device via an inlet. In these situations, the buffer solution is also applied to the device simultaneously and / or sequentially to the device via a second inlet to mobilize the binder and / or signal generator. Will be done.

In some practices, the method described herein is similar to the lateral flow bed / path in the lateral flow device described herein, for example, a test strip in which the plurality of scavengers are immobilized. Can be done with a test strip). The test strip may comprise a highly absorbent material in which the liquid sample and / or buffer flows through the strip under capillary force. When multiple targeted analytes are present in the liquid sample above certain detection thresholds (eg, clinically relevant thresholds), a detection complex can be formed and a detectable signal indicating a positive result. Can be provided. In the absence of the target analyte in the liquid sample, or in situations where the concentrations of the plurality of target analytes are below certain detectable thresholds, there are no detectable signals collected and / or observed. Shows a negative result.

In some practices, the method is the step of obtaining a liquid sample (eg, a blood and / or platelet sample, eg, a sample from a previously stored bag of blood and / or platelets that is potentially suitable for infusion). Can be included. In some practices, the sample may be further processed before being applied to the method and / or introduced into the devices of the present disclosure. The device (as described herein) can be used to identify the presence of bacterial, viral and / or fungal contamination in a liquid sample. In some practices, the detection and / or method may be performed prior to the use of blood and / or blood products in the infusion, thereby reducing the risk of infusing the contaminated blood product to the patient. In some practices, the sample is pretreated using base degradation followed by neutralization. In some practices, the pretreated liquid sample is optionally mixed with multiple binders prior to assay or introduction into the device. In some practices, the plurality of targeted analytes include bacterial, viral, and / or fungal antigens.

In some aspects, the present disclosure provides sequential lateral flow devices, such as devices for performing the methods described herein. The sequential lateral flow device described above is used to perform any of the methods described herein, including those described in the previous paragraph, to detect multiple target analytes. obtain.

In some embodiments, the sequential lateral flow device comprises a housing unit, the housing unit comprising an inner surface defining a cavity therein. In some practices, the sequential lateral flow device comprises a capillary flow bed in the cavity, where the capillary flow bed is from the proximal region of the capillary flow bed to the distal region of the capillary flow bed. It is configured to transport the above samples. In some embodiments, the sequential lateral flow device comprises a buffer receiving zone comprises a buffer receiving pad (P _B) and conjugates pads (P _C). The conjugate pad comprises a signal generator to provide a detectable signal. In some practices, the sequential lateral flow device comprises a capture zone (C) containing a plurality of immobilized capture agents. In some embodiments, the sequential lateral flow device comprises a sample receiving zone located between said buffer receiving zone and the capture zone, the sample receiving zone, the sample receiving pad (P _S), A transfer pad ( _PT ), as well as a substantially impermeable backing that is located between the sample receiving zone and the capillary flow bed and extends at least partially under the sample receiving pad and the transfer pad. Including. In some practices, the transfer pad comprises a plurality of binders that specifically bind to the plurality of target analytes. In some practices, the sequential lateral flow device comprises a reservoir pad (R) located in the distal region of the capillary flow bed. In some practices, the sequential lateral flow device includes a first entrance (I ₁ ) in the housing for introducing the sample into the sample receiving pad. In some practices, the impermeable backing inhibits contact between the sample and the capillary flow bed in the sample receiving zone, thereby causing backflow of the liquid sample in the proximal direction. Reduce. In some practices, the sequential lateral flow device includes a second inlet (I ₂ ) in the housing for introducing the buffer into the buffer receiving pad. In some practices, the buffer mobilizes the signal generator to obtain the mobilized signal generator. In some practices, the sequential lateral flow device is used to observe the detectable signal produced by the interaction of the plurality of binders with the signal generator in the presence of the target analyte. Includes a reading window defined in the housing unit beyond the capture zone. In some practices, the reservoir pad is adapted to pull the liquid sample and the mobilized signal generator in the distal flow direction. In some practices, the device is configured such that the sample flows along the capillary flow bed into the capture zone before the mobilized signal generator flows into the capture zone.

In some practices, the sample receiving zone, the buffer receiving zone, the trapping zone, and the reservoir pad are arranged on the capillary flow bed from the proximal region to the distal region as follows:

In some practices, the inner surface of the housing unit contains a series of ribs adapted to hold the excess fluid, whereby the excess fluid is the sample receiving pad, the buffer receiving pad, and /. Or it prevents the overflow from the above-mentioned capillary flow bed. For example, in such an implementation, if the user of the device adds an excess amount of the buffer reagent in the second port that exceeds the capacity of the buffer receiving pad, the ribs in the housing will Designed to contain the excess liquid. The same applies to the sample receiving pad and transfer pad described above.

In some practices, the series of ribs is placed on the sample receiving pad on the upper inner surface of the housing unit.

In some practices, the series of ribs is placed on the buffer receiving pad on the upper inner surface of the housing unit.

In some practices, the inner surface of the housing unit presses the buffer receiving pad against the conjugate pad, creating a flow path through which the buffer flows from the buffer receiving pad to the conjugate pad. Includes one pinch point.

In some practices, the inner surface of the housing unit presses the sample receiving pad against the transfer pad, creating at least one pinch point that creates a flow path through which the sample flows from the sample receiving pad to the transfer pad. Including.

In some practices, the inner surface of the housing unit comprises at least one pinch point where the transfer pad is pressed against the flow path bed and the sample makes a flow path from the transfer pad to the capillary flow bed.

In some practices, the capillary flow bed is slightly curved to promote capillarity and minimize overflow.

In some practices, the plurality of scavengers in the scavenging zone comprises one or more types of scavengers, each of which is at least a partial set of the target analyte in the liquid sample. It is adapted to specifically bind to a common antigen.

In some practices, the plurality of capture agents bind to Gram-positive and / or Gram-negative bacterial antigens.

In some practices, the plurality of capture agents include antibodies (eg, polyclonal antibodies, eg, multivalent polyclonal antibodies).

In some practices, the plurality of capture agents are selected from one or more types of polyclonal and monoclonal antibodies.

In some practices, the plurality of capture agents comprises one or more capture agent taxa on the capillary flow bed, the taxa being spatially separated from each other and each taxon. The group comprises antibodies that specifically bind to at least a partial set of different common antigens in the target analyte.

In some practices, the plurality of binders may bind to Gram-positive and / or Gram-negative bacterial antigens.

In some practices, the plurality of binders include antibodies. In some practices, the antibody is selected from one or more of polyclonal and / or monoclonal antibodies.

In some practices, each of the plurality of binders is associated with the first member of the conjugate pair and the signal generator is labeled with the second member of the conjugate pair.

In some practices, the conjugate pair is biotin and a biotin-binding protein, and either one of the plurality of binders or each of the plurality of signal generators is labeled with biotin.

In some practices, the signal generator is selected from the group consisting of metal particles, fluorescent dyes, and latex particles.

In some practices, the biotin-binding protein is selected from the group consisting of avidin, neutravidin, anti-biotin antibody, streptavidin, and other biotin-binding proteins.

In some practices, the capillary flow bed comprises nitrocellulose.

In some practices, the sample receiving pad, the buffer receiving pad, the reservoir pad, and the conjugate pad each contain a highly absorbent material.

In some practices, the highly absorbent material is selected from the group consisting of porous paper, polypropylene, polyester, polyethylene, glass fiber, cellulose blends, and combinations thereof.

In some practices, the sample is pretreated using base degradation followed by neutralization.

In some practices, the plurality of targeted analytes include bacterial, viral, and / or fungal antigens.

In some aspects, the disclosure provides a method for detecting multiple target analytes in a liquid sample using any of the lateral flow devices described herein.

In some practices, the step of detecting multiple target analytes in a liquid sample using any of the lateral flow devices described herein includes the following steps:
(A) The liquid sample can be introduced into the lateral flow device through the first inlet. The liquid sample flows from the sample receiving pad to the transfer pad, and the liquid sample is prevented from contacting the capillary flow bed by the backing before contacting the distal portion of the transfer pad. ..
(B) The plurality of target analytes, if present in the sample, form a first complex with the plurality of binders in the transfer pad. The first complex is pulled through the capillary flow bed towards the capture zone by the capillary force generated by the reservoir pad located in the distal region of the capillary flow bed.
(C) A buffer is introduced into the second inlet of the lateral flow device so that the mobilized signal generator flows through the visual window past the capture zone and then the capture. The signal generator may be mobilized after introducing the liquid sample to flow into the zone.
(D) The detectable signal generated in the capture zone can be read through the reading window.

In some practices, the sample may be added to the sample receiving pad using a dropper having a fixed volume / capacity calibrated for the capacity of the sample receiving pad. In some practices, a fixed amount of the liquid sample (eg, 45 μL to 50 μL of liquid sample) can be added to the sample receiving pad using the dropper, the fixed amount being the sample receiving pad. Greater than the saturated capacity of.

The present disclosure contemplates combinations of any of the above aspects, embodiments, and embodiments with each other, and with any one or more of the features presented herein.

This patent application includes at least one drawing with a color finish. A copy of this patent application gazette with color drawings will be provided by the authorities upon request and at the required fee.

The aforementioned and other objectives and advantages are apparent in light of the detailed description below, in conjunction with the accompanying drawings. Similar reference characters in the drawings refer to similar members throughout.

FIG. 1 shows a flow chart of the sequence of steps in a method for detecting multiple target analytes in a liquid sample, according to some practices.

FIG. 2 shows the formation of a sandwich complex via a two-step route according to some practices.

FIG. 3 shows a schematic view of the top surface of a sequential lateral flow device according to some practices.

FIG. 4 shows a schematic view of the capillary flow bed inside the cavity of the housing unit, according to some practices.

FIG. 5A shows a schematic diagram of a capillary flow bed according to some practices. FIG. 5B shows a schematic diagram showing the positioning of the reference point with reference to the capillary flow bed according to some practices.

FIG. 6 shows a schematic diagram of the configuration of the sample receiving zone according to some practices.

FIG. 7 shows a schematic view of the sides of the sample receiving zone according to some practices.

FIG. 8A shows a cut-out view of the housing alignment features of the friction-fit pin according to some practices. FIG. 8B shows each of the friction fit pins having a hard stop shape that prevents over-closure of the device during assembly, according to some practices. FIG. 8C shows that each of the friction fit pins is fully seated at the bottom of the cassette, according to some practices.

FIG. 9A shows a schematic diagram of a guide for the capillary flow bed and the reservoir pad (end core) reference according to some practices. FIG. 9B shows a schematic representation of the alignment tabs that maintain the position of the capillary flow bed relative to the housing features, according to some practices. FIG. 9C shows a schematic showing that, according to some practices, the alignment tab tilts away from the capillary flow bed, assisting the assembly and preventing overflow.

FIG. 10 shows a schematic representation of a liquid storage rib that acts as a liquid reservoir inside the upper portion of the housing unit, according to some practices.

FIG. 11 shows a photograph showing the curvature of the capillary flow bed inside the cavity of the housing unit, according to some practices.

FIG. 12A shows a schematic representation of a wide pinch contact point inside the housing unit that compresses the conjugate pad and directs the flow of liquid into the capillary flow path, according to some practices. FIG. 12B shows a schematic view showing the position of the cross-sectional view of the wide pinch point in FIG. 12C. FIG. 12C shows a cross-sectional view of the wide pinch point, at which the housing plastic tilts away from the contact point with the capillary flow bed, according to some practices. FIG. 12D shows a schematic cross-sectional view of the wide pinch point with reference to the sample receiving zone according to some practices. FIG. 12A shows a schematic representation of a wide pinch contact point inside the housing unit that compresses the conjugate pad and directs the flow of liquid into the capillary flow path, according to some practices. FIG. 12B shows a schematic view showing the position of the cross-sectional view of the wide pinch point in FIG. 12C. FIG. 12C shows a cross-sectional view of the wide pinch point, at which the housing plastic tilts away from the contact point with the capillary flow bed, according to some practices. FIG. 12D shows a schematic cross-sectional view of the wide pinch point with reference to the sample receiving zone according to some practices.

FIG. 13 shows a schematic diagram of the position of the pinch point with respect to the capillary flow bed according to some practices.

FIG. 14 shows a quantitative comparison of detection signals for detecting Pa 27853 at various dilutions using a non-sequential lateral flow device (direct method) vs. a sequential lateral flow device, according to some practices. ..

Detailed Description Unless otherwise defined herein, the scientific and technical terms used in this application shall have meanings commonly understood by those skilled in the art. The techniques and procedures described or referred to herein are well understood and are commonly used using conventional methodologies well known and commonly used in the art.

All publications, patents and published patent applications referred to in this application are specifically incorporated herein by reference.

Each implementation of the disclosure described herein may be adopted alone and / or in combination with one or more of the other implementations of the present disclosure.

Unless otherwise specified, the following definitions are provided with respect to the particular terms used in the description described above.

Throughout the disclosure, variations such as the terms "comprise" and / or "contain, comprises" and "contain, comprising" are the integers (or components) described. And / or the inclusion of a group of integers (or components and / or processes), but any other integer (or component and / or process) or group of integers (and / or component processes). It is understood that it does not mean the exclusion of.

The singular forms "one, are (a)", "one, are (an)", and "above, this, that (the)" include the plural, unless the circumstances specifically specify otherwise. .. "One, there (a)", "one, there (an)" also means "one or more" or "at least one".

Such as "include, include", "have", "include, contain", "involve", "composed of", etc. It should be understood that the transitional phrase is unrestricted and is used to mean "including, but not limited to (inclusion but not limited to)". "Including" and "including, but not limited to," are used interchangeably.

As used herein, a binder is an agent capable of binding more than one genus of bacteria, viruses, and / or fungi. Binders, when used in the methods and devices of the present disclosure, have more than one genus of bacteria, viruses, and / or fungi (eg, two or more of bacteria, viruses, and / or fungi, 3). Or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more More, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more , Or 20 or more genera) common antigens. In some practices, the binder is an antibody. In some practices, multiple binders are used in the methods and devices described herein. The plurality of binders can be one or more types of binders (eg, one or more types of antibodies), each of which is a common antigen of at least a partial set of the target analyte. Suitable for binding specifically to, the target analyte may comprise a large number of subsets or types of analyte, each subset or type having a common antigen within that subset. In some practices, the binder specifically binds to a common antigen in more than one genus of bacteria, viruses, and / or fungi. According to a non-limiting example, an antibody that specifically binds to lipopolysaccharides of two or more genera of Gram-negative bacteria is a binder. Similarly, an antibody that specifically binds to lipoteichoic acid (LTA) in two or more genera of Gram-positive bacteria is a binder. Such binders can be polyclonal antibodies and / or monoclonal antibodies. In some practices, the binder comprises an antibody having various specificities in the mixture such that the mixture binds to more than one genus of bacteria, viruses, and / or fungi. Other non-antibody molecules can act as binding agents if they have the ability to bind to bacterial, viral, and / or fungal components (eg, antibiotics such as polymyxin are the majority of Gram-negative bacteria). It binds to lipopolysaccharides of the genus, and vancomycin can bind to components of the cell wall of Gram-positive bacteria). These molecules can be used as binders with suitable linkers.

As used herein, an "antigen" (eg, a Gram-negative bacterial antigen and / or a Gram-positive bacterial antigen) is in any structural conformation that can be specifically bound by a binding agent. Used to mean any molecule. The site on the antigen that is bound by the binder is referred to as the "binding site". Antigens can be, but are not limited to, proteins, glycoproteins, carbohydrates, and / or lipids.

As used herein, "analytes" or "analytes" are species, substances, and / or species that should be detected and / or quantitatively analyzed in a sample. Refers to a compound. Analysts include, but are not limited to: toxins, proteins, peptides, viruses, bacteria and / or bacterial antigens, nucleic acids, carbohydrates, fungi, steroids, hormones, polysaccharides, carbohydrates, contaminants, Metabolites, antibodies, and / or any detectable substance from human and / or non-human sources (eg, blood, tissues, water, soil, sewage, beverages). In some practices of the present disclosure, the analyte bound to the binder to form a first complex, which was then immobilized on a capillary flow bed in its scavenging zone. It binds to the scavenger and forms a sandwich complex between the first complex and the scavenger. In certain such practices, the sandwich complex further binds to the signal generator to form a detection complex. The formation of each complex is separate and sequential.

As used herein, a "conjugation pair" or a "conjugate pair" is one in which the first and second molecules / members are covalently bonded, compatible. , And / or two different molecules / members that are attached to each other through physical means. The binding or interaction between the members of the conjugate pair is due to the various interactions and / or affinities that the member derives from the other components and / or surrounding substances of the assay. It is specific and peculiar so that it can be distinguished. In some practices, the conjugate pair can be a receptor and a ligand (eg, an antibody and an antigen). In other practices, conjugate pairs include, but are not limited to: biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and Enzymes, enzyme inhibitors and enzymes, peptides sequences and antibodies specific for the sequence or the entire protein, polymer acids and bases, dyes and protein binding agents, peptides and specific protein binding agents (eg, ribonuclease, S-peptide, and ribonuclease). S-proteins), metals and their chelating agents, etc. In addition, the specific conjugate pair is a member that is an analog and / or derivative of the original specific binding member (eg, a chemical modification, recombination technique, and a recombination technique that still maintains binding properties similar to the other binding member. / Or specific binding members made by molecular manipulation). In some practices, the first member of the conjugate pair is biotin and the second member of the conjugate pair is selected from avidin, neutravidin, streptavidin, and / or any anti-biotin antibody. Will be done.

The present disclosure provides methods, sequential lateral flow devices, and kits for detecting one or more target analytes in a liquid sample. The disclosure also provides a method of using the disclosed device and / or kit. It should be understood that the present disclosure is not limited to the practices set forth herein. It should also be understood that the implementation of this disclosure is intended for explanatory purposes and should not be considered as a limitation.

In some practices, the present disclosure provides methods, devices and kits that have broader reactivity and higher sensitivity than existing methods, devices and kits. In some practices, the methods, devices, and kits may detect a wider range of targeted analytes (eg, a wider range of bacterial genera, species, and / or bacterial strains than existing methods and devices). .. For example, the methods, devices, and / or kits are at least 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, Or 500 different bacteria, viruses, or fungi can be detected. In some practices, the present disclosure is 1x10 ⁷ , 1x10 ⁶ , 1x10 ⁵ , 1x10 ⁴ , 1x10 ³ , or more than 1x10 ² colony forming units (CFU) / mL. Provided are methods, devices, and / or kits comprising multiple antibodies capable of detecting the bacterium and / or an equal concentration of antigen obtained from that level of the bacterium, each of which is one of the above targeted analysts. It can bind at least a partial set of common antigens.

A detailed description of certain practices appropriate for the devices, kits, and methods of the present disclosure is considered, but is not limited to:

FIG. 1 shows an exemplary method (100) for detecting multiple target analytes in a liquid sample according to some practices. In step (a), the liquid sample is contacted with a plurality of binders and a plurality of scavengers on a solid support in a lateral flow device under conditions that allow the formation of at least one sandwich complex. .. The sandwich complex is composed of one or more of the multiple binders and one or more of its target analytes and one or more of its scavengers. Formed between. The sandwich complex can be formed in a stepwise fashion (eg, the target analyte can first bind to the binder before binding to the scavenger, or the target analyte can bind to the binder. Can first bind to its scavenger before binding to). In some events, the target analyte, binder, and scavenger thereof can be combined to form the sandwich complex in one step. In some practices, the plurality of binders and the plurality of scavengers are antibodies. In some practices, the plurality of binders and the plurality of capture agents thereof are monoclonal antibodies, polyclonal antibodies, and / or mixtures thereof. In some practices, the plurality of binders and the plurality of capture agents are polyclonal antibodies. The sandwich complex comprises at least one target analyte (in the center), one or more of its scavengers (at the bottom immobilized on a solid support), and multiple binders thereof. Includes one or more of them (at the top). Unbound reagents, target analytes, and / or binders, if necessary, wash the solid support with buffer and / or centrifuge the solid support prior to the next step, for example. Can be washed away and / or removed by drying. When the method is performed on a device (eg, a lateral flow device as described herein), the unbound reagent can be carried away from the scavenger via capillary force.

In some practices, the plurality of binders and / or the plurality of capture agents thereof are antibodies (eg, polyclonal or monoclonal antibodies). In some practices, such antibodies may specifically bind to at least a partial set of Gram-positive and / or Gram-negative bacteria and / or at least a common antigen of that subtype. In certain practices, the plurality of binders or capture agents thereof may be a polyclonal antibody (eg, a polyvalent polyclonal antibody), a monoclonal antibody, and / or a mixture thereof. In some practices, each of the multiple binding agents is a conjugate pair (eg, biotin and biotin-binding proteins (eg, avidin, neutravidin, antibiotin antibodies, streptavidin, and / or other biotin-binding proteins). It is tagged with one member of). Unlike the binder-signal-producing particle conjugates used in conventional methods, the binder in the methods of the invention is, according to some practices, a low first member of the conjugate pair (eg, biotin). It is tagged with a molecule). Thus, the size of the binder is much smaller than that of the binder-signal-producing particle conjugate. Within that same reaction space, the local concentration of binder can be greatly increased, thereby increasing the number of its first complex for the next step and finally for detection. Produces an increased binding event to produce. In some practices, the binder is labeled with biotin. In some practices, the binder is labeled with a biotin-binding protein. Suitable conditions that may facilitate the formation of the sandwich complex are to carry out the reaction in a buffer solution (eg, phosphate buffer) at room temperature, optionally with stirring and / or slight heating. Can be mentioned. If desired, the unbound target analyte or binder can be removed prior to the next step.

In some practices, the plurality of scavengers can be one or more types of scavengers, each to a common antigen in at least a partial set of the target analyte in its liquid sample. It is adapted to be specifically bound. For example, in some practices, the scavenger may specifically bind to at least a partial set of Gram-positive and / or Gram-negative bacteria and / or at least a common antigen of that subtype. In some practices, the capture agent is an antibody, eg, a polyclonal antibody (eg, a polyvalent polyclonal antibody), a monoclonal antibody, and / or a mixture of those described above. In some practices, at least some of the scavengers are of the same type as at least some of the binders. In some practices, at least some of the scavengers are of a different type than at least some of the binders. In some practices, the multiple capture agents are covalently bonded and / or physically absorbed through solid support (eg, test strips and / or capillary flow beds for the devices described herein). ) It is fixed in the above classification group (group). The taxa may be spatially separated from each other, and each taxon contains an antibody that specifically binds to a different target analyte.

The process continues to step (b), where the signal generator (eg, non-enzymatic) is subject to conditions that allow the signal generator to bind to the binder of the sandwich complex. Contact with the sandwich complex forms a detection complex. The signal-generating agent is tagged with a second member of the conjugate pair, and the binding of the signal-generating agent to the binder of the sandwich complex is of a plurality of target analytes in the liquid sample. Indicates the presence of one or more species. Thus, the detection complex is also immobilized on the solid support in which the scavenger is present. The lateral flow device is adapted to inhibit the signal-generating agent from contact with the plurality of binders prior to the formation of the sandwich complex. In some practices, the lateral flow device has a sample receiving pad in the sample receiving zone for introducing the liquid sample into the lateral flow device and a capillary flow in the solid support that facilitates the flow of the liquid sample. It contains a virtually opaque backing placed between the bed and thereby reducing the backflow of the liquid sample in the proximal direction of the lateral flow device. In some practices, the liquid sample may be added to the sample receiving pad using a dropper having a fixed volume / capacity calibrated to the saturation capacity of the sample receiving pad. In some practices, the amount of liquid sample added by using the dropper is less than the fully saturated capacity of the sample receiving pad so that the sample receiving pad can be in an unsaturated state.

The signal generator is detectable, in which the detection complex is formed on the solid support to indicate the presence of one or more of the plurality of target analytes in the liquid sample. Provide a signal. In some practices, the signal generator is pigmented particles, latex particles, metal particles (eg, gold, silver, platinum nanoparticles), fluorescent particles, or magnetic particles. In some practices, the signal generator is a colored dye and / or a fluorescent dye. In some practices, the signal generator is a catalytic enzyme. In some practices, the signal generator is a particle (eg, gold nanoparticles (eg, 40 nm, 60 nm, or 80 nm gold nanoparticles). In certain such practices, the gold nanoparticles are , Tagged with the second member of the conjugate pair, and the binding of the signal-generating agent to the binder of the sandwich complex (eg, via the conjugate pair) is a plurality of targets in the liquid sample. It shows the presence of one or more of the analytes. In certain such practices, the signal generator is tagged with a biotin-binding molecule (eg, avidin and / or streptavidin). Gold nanoparticles. The excess signal generator can be washed away and / or removed prior to signal detection to minimize interference.

The process continues to step (c), where the signal produced by the formation of the detection complex determines the presence of one or more of the multiple target analytes in the liquid sample. To be detected. The signal can be detected using appropriate means (eg, visual, electrical, and / or optical detection). If one or more of the target analytes are present in the liquid and the detection complex is formed in sufficient quantity, the signal will be signaled, for example, on a solid support on which the scavenger is immobilized. Detected and gives a positive result. However, in the absence of one or more target analytes, or in the absence of one or more target analytes at clinically relevant levels, the first complex. Neither the sandwich complex nor the detection complex is formed in sufficient amounts, so no detectable signal is found, which results in a negative result.

FIG. 2 shows two pathways for forming a sandwich complex. Each route has two steps that follow some practices. In the first pathway (200), the liquid sample forms a first complex between the plurality of binders and at least some of the target analytes and at least some of the binders. Are contacted under conditions that allow for. The first complex is then contacted with the plurality of scavengers to form the sandwich complex such that the liquid sample contacts the plurality of scavengers after the formation of the first complex. To do. In operation, the liquid sample may simply be mixed with the plurality of binders in a test tube before flowing through the devices described herein. The solution containing the first complex is also immobilized on the solid support (on the solid support, the scavenger is immobilized by immersing the solid support in the solution for a sufficient amount of time). Can be contacted by. Alternatively, the liquid sample is preloaded with the plurality of binders propelled by tensile forces (eg, capillarity), devices (eg, lateral flow devices (eg, devices described herein)). It can flow through and / or on test strips (eg, lateral flow beds / paths used in the devices described herein). The device or strip is configured such that the liquid sample is in contact with a plurality of binders before being contacted with the plurality of capture agents in the capture zone. Appropriate conditions can be applied to ensure complex formation between the target analyte and its binder and / or its capture agent. For example, in the first pathway (200), this first step takes time to allow the first complex and / or binding so that the formation of the sandwich complex can be as complete as possible. It is performed at room temperature and / or with slight heating, if necessary, provided that the untargeted analyte and binder can interact with the plurality of capture agents.

In the second route (300), the liquid sample is placed between a plurality of scavengers placed on a solid support in a lateral flow device or test strip and between one target analyte and one scavenger. The liquid samples are then contacted with the plurality of binders to form the sandwich complex. In practice, the liquid sample may first flow through the lateral flow device, followed by a separate flow of buffer containing the multiple binders.

According to one aspect, the sequential lateral flow device of the present disclosure includes a housing unit, the housing unit having an inner surface defining a cavity therein.

3 and 4, respectively, show a top view and an inside view of the sequential lateral flow device according to some practices. As shown in FIG. 3, the housing unit may include an upper portion (10) that may fit together with a lower portion (11) (FIG. 4) to form the cavity. The upper part of the housing unit is the first entrance (20) (some of which are shown in red and are marked with a "1" and a red outline in FIG. 3), the second entrance (21) (some of which entrances are shown in white and are represented by "2" in FIG. 3), and may include a reading window (22). The liquid sample can be introduced into the device through its first inlet (20), while the reagent buffer can be introduced into the device through its second inlet (21).

FIG. 4 shows a capillary flow bed (30) inside the cavity of its housing unit, according to some practices. In some practices, the capillary flow bed has a proximal region and a distal region. In some practices, the capillary flow bed is secured to the lower portion (11) of the housing unit, which, as shown in FIG. 4, has a restricted contact point with the capillary flow bed (eg, for example. Have (through a discontinuous and separate support under its capillary flow bed).

In some practices, the housing unit maintains its capillary flow bed inside the housing unit, eg, in the lower portion of the housing unit, properly aligned and anchored inside. It may further include features such as criteria and alignment tabs. In some implementations, the housing unit, such as ribs, discontinuous supports, and / or pinch points, to control the flow of liquid through its capillary flow bed, eg, in a sequential fashion. Features can be further included inside. In one aspect, the capillary flow bed is configured to transport a liquid (eg, sample and reagent buffer) from the proximal region to the distal region of the capillary flow bed through capillarity. Further features of the capillary flow bed are shown herein using similar numbers as shown in FIG. 4, according to some practices.

FIG. 5a shows the capillary flow bed and the various components on the bed according to some practices. In some practices, the capillary flow bed may include the following zones: buffer receiving zone, sample receiving zone, and capture zone. Its buffer receiving zone is disposed on the capillary flow bed, including buffer receiving pad _(P B, 40) and conjugate pad _(P C, 41). The conjugate pad is placed on the conjugate pad (eg, attached to the conjugate pad and / or associated with the conjugate pad) to provide a detectable signal. It contains a product (eg, gold nanoparticles labeled with biotin-binding protein). The signal generator can be released and dried inside the conjugate pad (41). The reagent buffer has its second entrance (to its buffer receiving pad) to mobilize the signal generator retained in its conjugate pad to flow through its capillary bed into its capture zone. It can be introduced through I2, 21). The capture zone (C, 44) is a plurality of capture agents (eg, one of the capture agents or multiple types of capture agents greater than one) (eg, on the capillary flow bed (eg, 1). Includes (in a species or more than one taxon) an antibody that specifically binds to a common antigen in a subset of its multiple target analytes). In some practices, the sequential lateral flow device may further include a reservoir pad (R, 45) located in the distal region of the capillary flow bed (30). The reservoir pad is adapted to pull the sample and buffer and carry the mobilized signal generator in the distal flow direction. In some practices, the sample receiving zone is located between the buffer receiving zone and the trapping zone. The sample receiving zone includes a sample receiving pad (Ps, 42) and a transfer pad (Pt, 43). According to some practices, the sample receiving zone also contains an impermeable backing (60). FIG. 5b shows the position of the reference point with reference to the capillary flow bed.

In some practices, the devices of the present disclosure include capillary flow beds (eg, such as those shown in FIGS. 5a and 5b). FIG. 5a shows a capillary flow bed with various components on its bed, according to some practices. In certain such practices, sample receiving zones (including sample receiving pads (42) and transfer pads (43)), buffer receiving zones (buffer receiving pads (40) and conjugate pads (41)), The reservoir pad (45) is also arranged on its capillary flow bed (30).

In some practices, the capillary flow bed (30) and / or its sample receiving pad (42), and / or its transfer pad (43), and / or its buffer receiving pad (40), and / or The conjugate pad (41) can be made from a highly absorbent material (eg, a nitrocellulose membrane). The highly absorbent material of the present disclosure is a porous material having pores, which can transport a liquid through the membrane according to the capillary force. The highly absorbent material comprises a series of fibers that are pulled together in parallel to form an open core. The spaces between the fibers form channels that pull the liquid through capillarity.

In general, highly absorbent materials are hydrophilic in nature. Suitable highly absorbent materials include, but are not limited to: hydrophilic inorganic powders (eg, silica gel, alumina, diatomaceous earth, etc.), sponge substances, glass fibers, clayey substances, cloth, hydrophilic. Natural polymeric materials of sex, especially cellulose materials, similar cellulose beads, and especially fiber-containing papers (eg, filter paper and / or chromatographic paper), synthetic and / or modified naturally occurring polymers (eg, eg). Nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, polyethylene, glass fiber, polyacrylate, polyurethane, crosslinked dextran, agarose, and other such crosslinked and uncrosslinked water-insoluble hydrophilic polymers). Suitable highly absorbable materials of the present disclosure can be functionalized on the surface and form covalent bonds with, for example, antibodies and / or receptors.

In some practices, the highly absorbent material can be in the form of pads, sheets, and / or compressed fibers. In some practices, the highly absorbent material is nitrocellulose (eg, nitrocellulose with pore sizes of about 0.4 microns to about 15 microns).

In some practices, the capillary flow bed is a solid support (eg, a substantially impermeable support), including a water-insoluble, non-porous, flexible plastic strip (eg, a polyester strip). Placed on top. In general, the solid support is of the same or slightly different dimensions as the capillary flow bed. Suitable materials for the solid support include polyethylene, polypropylene, polystyrene, polymethacrylate, nylon, glass, ceramic, metal, polyurethane, neoprene, latex, silicone rubber, polyester, poly (ethylene terephthalate), poly (vinyl butyrate). ), But not limited to these.

In some practices, the capillary flow bed may include at least one capture zone (44), as shown in FIGS. 5a and 5b. The capture zone comprises one or more capture agents that are immobilized on the capillary flow bed via chemical and / or physical means.

In some practices, when the liquid sample is added to the sample receiving pad (42), the sample flows under capillary force to the transfer pad (43) and mixes with its multiple binders. Then, a first complex is formed between at least some of the binders and some of the target analytes. The sample with the first complex continues to flow through its capture zone through its capillary flow bed. The plurality of capture agents then bind to the target analyte and form a sandwich complex between the capture agent and the target analyte, the binder. The signal-producing agent then binds to the binder of the sandwich complex through its conjugate pair as the buffer containing the signal-generating agent flows through its scavenging zone, and with the signal-generating agent. , The binder, the analyte, and the capture agent to form a detection complex. In some practices, the first complex is in contact with the plurality of scavengers so that the liquid sample is in contact with the plurality of scavengers only after the formation of the first complex. Form a sandwich complex. The signal-producing agent retained thereby provides a detectable signal, which indicates the presence of one or more of the plurality of target analytes in the sample. Thus, the flow of liquid is tightly controlled in a sequential fashion, and the devices of the present disclosure are thus designed to ensure a sequential and gradual flow of liquid through its capillary bed.

In some aspects, the various features of the capillary flow bed described herein are that of a fluid that traverses the capillary flow bed (30) and passes through an immobilized scavenger in its capture zone (44). It can be incorporated to ensure sequential flow and minimize fluid mixing at their interfaces. The sensitivity of the devices of the present disclosure can result from a combination of factors discussed herein, in particular the sequential flow of fluid and the minimal mixing of liquids. In the present disclosure, the liquid is (1) a liquid sample mixed with a plurality of binders (eg, an antibody (eg, polyclonal antibody)); or (2) a signal generator (eg, streptavidin-labeled gold nanoparticles). References to buffers with.

In some practices, the capillary flow bed comprises a control zone, wherein the signal-generating agent exhibits its proper functionality and / or as a means of confirming the effectiveness of the method performed. It is captured and retained even in the absence of the analyte. In some practices, the control zone comprises an immobilized member of the conjugate pair (eg, biotin), which member binds to the signal generator (eg, streptavidin-labeled gold nanoparticles). Can be. In some practices, the control zone may be located behind the capture zone on the capillary flow in its distal region, but in front of its reservoir pad.

In some practices, the capillary flow bed comprises two fluid addition zones, namely a sample receiving zone and a buffer receiving zone, as shown in FIGS. 5a and 5b. In some practices, the sample receiving zone comprises a sample receiving pad (42) and a transfer pad (43). In some practices, the buffer receiving zone comprises a buffer receiving pad (40) and a conjugate pad (41).

As shown in FIGS. 5a and 5b, the sample receiving zone is located in the center of the capillary flow bed between the buffer receiving zone and its trapping zone (45), according to some practices (eg, its). It is placed (in the center of the lower third of the capillary bed).

6, according to some embodiments, showing an example of a sample receiving zone comprising a sample receiving pad _(P S, 42) and transfer pad _(P T, 43). The sample receiving zone of FIG. 6 can be used in any of the devices and / or methods disclosed herein. As shown in FIG. 6, a substantially opaque backing (60) is located between the sample receiving zone and its capillary flow bed, even if not under the sample receiving pad and its transfer pad. It is partially extended. In some practices, if the sample is introduced into the sample receiving pad through its _first entrance (I ₁ , 20), its impermeable backing (60) is that sample and its. It inhibits contact with the capillary flow bed in the sample receiving zone, thereby reducing the backflow of the sample in the proximal direction. In some practices, the plurality of binders (eg, antibodies (eg, biotin-labeled polyclonal antibodies)) may be placed inside the transfer pad. In some practices, the binder can be dried inside its transfer pad and bind to at least a partial set of common antigens in the target analyte. In some practices, the binder is contained within the sample receiving pad.

In some practices, the sequential lateral flow device has its liquid sample along its capillary flow bed (30) before its mobilized signal generator flows into its capture zone (45). It is configured to flow into the capture zone (45), and its usage is made. In some practices, upon operation, the reading window (22) of the sequential lateral flow device is detectable due to the interaction of the binder with the signal generator in the presence of the target analyte. It is placed above its capture zone (44) to observe the signal. The construction of the sample receiving zone is optimized to control and minimize the backward flow of the sample mixed with the binder (eg, antibody, eg, biotinylated antibody). In some practices, the transfer pad (43) is reconstituted and mobilized as a dry binder (eg,) as the sample is added to the sample receiving pad through the first inlet (20). , Antibodies). In some practices, the sample pad is substantially non-existent in order to minimize and control the posterior flow of the first complex between the sample and / or the sample and the binder. It remains on a permeable plastic backing (60) and acts as a barrier between most of its transfer pad and its underlying capillary flow bed (the capillary flow bed contains a nitrocellulose pad). .. The only contact that the transfer pad has with the capillary flow bed is through the anterior lip of the transfer pad. It is only through this contact that any fluid flows from its transfer pad onto its capillary flow bed.

FIG. 7 shows a schematic side view of the sample receiving zone inside the housing unit according to some practices. As shown in FIG. 7, the transfer pad is curved at the anterior tip to contact the capillary flow bed due to the impervious backing (60) underneath, according to some practices. Can be done.

In some practices, when the sample is added, the fluid flows from the sample receiving pad through the transfer pad onto the capillary flow bed. This fluid then flows forward towards its capture zone and backward towards its conjugate pad.

In some practices, the buffer receiving zone comprises a buffer receiving pad (40) and a conjugate pad (41), as shown in FIGS. 4a and 4b. The buffer receiving pad and its conjugate pad each contain the same or different types of highly absorbent materials. Suitable buffer solutions to be used in the present disclosure include, but are not limited to: acetate buffers, buffered physiological saline, citrate buffers, barbital buffers, phosphate buffers, or Tris ( Hydroxyl-methyl) aminomethane (TRIS), N- (2-acetamide) iminodiacetic acid (ADA), piperazin-1,4-bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamide)- 2-Aminoethanesulfonic acid (ACES), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N-tris (hydroxymethyl) methyl -2-Aminoethanesulfonic acid (TES), 2,4- (2-hydroxyethyl) -1-piperazinyl Etansulfonic acid (HEPES), 3,4- (2-hydroxyethyl) -1-piperazinyl Propane sulfonic acid (EPPS), N-tris (hydroxymethyl) methylglycine (tricin), N, N-bis (2-hydroxyethyl) glycine (bisin), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), Buffer solution prepared with N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) and / or K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , NaHCO ₃ , NaBO ₄ , ( NH4) A buffer solution containing one or more buffer salts selected from ₂ CO ₃ .

After the liquid sample is added through the first inlet (20), the reagent buffer (eg, chase buffer, eg, phosphate buffer) is introduced to the device at its second inlet (21). It can be introduced through and onto the buffer receiving pad (40) at the distal upstream end of its capillary flow bed. Avoid situations where binders (eg, biotinylated antibodies) encounter and bind to their signal generators (eg, streptavidin-conjugated gold nanoparticles) in excess of a small amount at their fluid interface. And to prevent the sample fluid from flowing back into its conjugate pad, the buffer will flow once the front of the sample fluid has flowed past its confirmation window beyond its capture zone. Can be added immediately. The timing of adding the buffer ensures that there is a countercurrent flow of the forward fluid against the sample mixture flowing backwards. As a result, these two fluids meet in a very small volume defined by the pore size of the nitrocellulose pad of the capillary flow bed. Therefore, almost no mixing occurs between these two types of fluids.

The forward flow of the sample reaches the reservoir pad (45), which acts as a sump that pulls the fluid onto the reservoir pad itself. As long as this forward flow is imposed by the reservoir pad, there is little mixing at their interface between the two fluids and their sequential flow is maintained. In some practices, in approximately 5-7 minutes, the flow of the sample fluid should be complete and the anterior part of the flow of liquid containing the signal generator should begin to appear in its reading window.

In some practices, the devices of the present disclosure include a reservoir pad (45). In some practices, the reservoir pad is placed in the distal region of the capillary flow bed, eg, at the end of the distal region of the capillary flow bed. In some practices, the reservoir pad also contains a highly absorbent material (eg, a nitrocellulose pad), which acts as a liquid sink, combining its capillary flow bed, sample receiving zone, and buffer receiving zone. It has a liquid absorption capacity that exceeds the total capacity. In this way, during the process of performing the method, a continuous capillary force is generated across the capillary flow bed from the proximal region to the distal region. This continuous capillary force is important to maintain its flow in one direction and reduce the risk of backflow and / or fluid mixing.

In some aspects, there are many considerations for designing a housing unit (manufacturability of the device, tolerances of its components, component size (eg, component assembly and placement), The functionality of the assay (including, for example, the variability of how the assay is performed due to user-related factors), and the overall aesthetics). In some practices, the device components may be secured to the housing unit so that the various components of the device are properly aligned with respect to the inlet (or liquid reagent well) and reading window. For example, as shown in FIGS. 2 and 3, the capture zone is provided in the reading window and the housing unit does not interfere with testing using the methods disclosed herein and / or test results. Do not change the quality. Various device components include, but are not limited to, capillary flow beds, buffer receiving zones, capture zones, sample receiving zones, and reservoir pads to enable detection methods. In other practices, the housing unit may include features to facilitate control and sequential flow of liquids (eg, sample and reagent buffers).

In some practices, the dimensions of the housing are partly defined by the requirement that the device be read by off-the-shelf lateral flow assay (LFA) readers, if desired. .. Such an LFA reader (eg, Qiagen ESEQuant LR3) defines its device housing to be of some size (eg, smaller than about 110 mm in length, about 50 mm in width and about 12.5 mm in height). Can be done. In addition to the LFA reader considerations, the structure of the capillary flow bed with the various components of the device also affects the overall housing dimensions. In some practices, the length of the capillary flow bed is at least about 80 mm, at least about 90 mm, to facilitate the sequential flow of liquid and adapt the liquid volume along all the other components of the device. Should be at least about 100 mm, or at least about 110 mm. In some practices, the length of the capillary flow bed can be in the range of about 50 mm to about 200 mm, about 55 mm to about 180 mm, about 60 mm to about 160 mm, about 75 mm to about 140 mm, or about 80 mm to about 100 mm. .. In some practices, the capillary flow bed has a length of about 85 mm. However, the housing can be of any suitable shape and / or size, including sizes that are not compatible with automated assay readers.

3 and 4 show a top view and an inside view of the housing unit according to some practices. In some embodiments, the housing unit includes an upper portion (10) paired with a lower portion (11). In some such practices, the upper portion of the housing unit includes a reading window (22). In some practices, the upper portion of the housing unit further comprises a first inlet (20), eg, a first inlet for introducing the sample into the sample receiving pad (42). In some practices, the upper portion of the housing unit further comprises a second inlet (21), eg, a second inlet for introducing the buffer solution into the buffer receiving pad (40).

In some implementations, the reading window clearly distinguishes the control line from the other capture zones so that the user can interpret the test results accurately so that the control lines from other capture zones are clearly distinguished. Designed and arranged to identify and separate.

FIG. 3 shows that the reading window narrows at the distal end (eg, at the position of the control line) according to some practices. In some such practices, the reading window may be narrowed from about 5 mm to about 3 mm. Narrowing provides a physical and visual distinction that intuitively warns the user that the control line is distinguished from other capture lines. In some practices, the lead window has a slope on the side wall away from the strip surface to minimize shadow projection, which can make it more difficult to properly visualize positive results.

In some practices, the reading window has a length of about 10-50 mm, about 20-40 mm, or about 20 mm, about 30 mm, about 40 mm, or about 50 mm. In certain such practices, the reading window has a length of about 23 mm.

In some practices, the width of the reading window is smaller than the width of the capillary flow bed. In certain such practices, the width of the reading window is about 2 mm to 10 mm, about 4 mm to 8 mm, about 5 mm to 6 mm, or about 3 mm, about 4 mm, about 5 mm, or about 6 mm. In some practices, the width of the reading window is about 4.7 mm. In other embodiments, the width of the reading window is about 2.6 mm.

FIG. 3 shows that, according to some practices, the upper portion of the housing (10) has a flat area on the top surface for labeling, eg, for bar code stickers. In some practices, the upper portion of the housing unit comprises marking the top surface thereof to identify where the samples and reagents should be added. In some practices, the upper portion of the housing unit has a recessed surface around its first and second inlets to form a liquid reservoir, which is a disposable pipette and Facilitates the transfer of liquid in the dropper bottle.

In some practices, the housing unit is designed to be easy to manufacture. For example, the upper and lower ends of the housing unit may be parallel to the short portion to assist in automated sorting and alignment of the upper and lower portions thereof. In addition, the upper portion of the housing unit is modified so that a standard roller-closure can be utilized to assemble the device.

In some embodiments, the upper portion of the housing unit comprises at least one friction fit pin on its inner surface. 8a-8c show friction fit pins with alignment guide elements according to some practices. In some such practices, the area under the friction fit pin is substantially flattened to allow uniform pressure on the pin, which is important for device closure. In some practices, as shown in FIG. 8a, the friction fit pin allows the upper and lower parts of the housing unit to be approximately aligned and "honed in" during pairing. As you can get, it is conical. In some embodiments, the upper portion of the housing unit is one or more friction fit pins (eg, 1, 2, 3, 4, 5, 6, 7, 7, 8). , 9, or 10 friction fit pins).

8b and 8c show, according to some practices, in addition to incorporating alignment features into the friction fit pins, each of the friction fit pins is a hard stop that prevents overclosure of the device during assembly. Showing additional features, the hard stop features can potentially damage the capillary flow bed.

The design strategy for its press-fit features further requires considerations for many other features that are not easily apparent from structural references, one such consideration creating an injection molded housing unit. It is a limitation of the readily available tool makers used for. The tool maker may have the limitation that the mold cannot be reasonably reworked in order to make what is known as a "steel-safe" part. In some situations, the tool manufacturer selected for its industry-leading rapid prototyping of injection molded parts cannot rework the tool to tolerances of less than 0.003 inches. Due to this limitation, the tool manufacturer cannot guarantee snap-fit and / or press-fit features. To address this limitation, in some practices, the friction fit feature incorporates a pin that deforms upon closure. In other practices, the female component of the component can also be deformed to accommodate the pin. By designing its press-fit feature as a deformable element, the acceptable tightening tolerance is increased to about 0.005 inch, which is within the tolerance of its traditional distributor's tool making capabilities.

One aspect of the housing design is the need to properly secure the capillary flow bed and each component to the top of the housing in its desired position. Figures 9a-9c show the alignment tabs and references as well as their position on the inner surface of the housing unit, according to some practices. FIG. 9a shows that the capillary flow bed and its housing unit were located in the distal region of the capillary flow bed (eg, at the end of the reservoir pad (or end core) of the capillary flow bed, according to some practices. Show that common criteria can be referred to. It is from this common standard that the tolerances of the capillary flow bed and its housing unit are derived in certain such practices. In some practices, the reference for the housing unit is the tab on which the distal region of the capillary flow bed is pushed.

FIG. 9b shows alignment tabs on the inner surface of the housing unit to maintain the position of the capillary flow bed according to some practices. In certain such practices, in addition to the terminal core reference point tab (terminal-wick-datum-point-tab) in the housing unit, the devices of the present disclosure make the capillary flow bed suitable for the housing unit. It further includes a set of alignment tabs (eg, 2 or more alignment tabs, eg, 2, 4, 6, or 8 alignment tabs) that are aligned to the features. These alignment tabs place the capillary flow bed at an important pinch point at its first and second entrances (ie, sample and buffer wells) and at the interface between its housing unit and its capillary flow bed. And collect and maintain.

FIG. 9c shows that, according to some practices, the alignment tab tilts away from its capillary flow bed to assist the assembly and prevent overflow. In certain such practices, the capillary flow bed and its components can be properly placed in any case, either manually and / or by automation. To make this task easier, the alignment tabs are tilted to help guide the capillary flow bed to the proper position. In some cases, the housing may provide a potential wetting point and may unintentionally spill out of the interior and / or cavity of the housing unit. In some practices, to avoid unintended overflow-like situations, the alignment tabs have their capillary flow beds so that their liquid reagents are contained within the boundaries of their pads and the features of their housing units. Can tilt positively away from the surface of the.

In some aspects, the methods and devices of the present disclosure are designed to detect a large number of targeted analytes (eg, bacterial antigens from a wide variety of bacterial species and strains). In some practices, the methods and devices of the present disclosure may use a large number of complex antibody mixtures. In some practices, multiple capture zones of the antibody can be immobilized on a capillary flow bed (eg, a nitrocellulose pad) in the devices of the present disclosure. A wide variety of antigen targets must be present in an amount sufficient to label the antigen sufficient to produce an equally diverse and detectable signal, in order to facilitate detection. It means not to be. The better performance of using the devices of the present disclosure is to include the labeled antibody, so that the sample traverses its capture zone, and then its detection component (eg, a signal generator (eg, streptavidin)). It is determined that this is achieved by sequentially flushing the conjugate colloidal gold particles)). The specific need to sequentially flow the sample and reagent (eg, buffer solution) through the device in a controlled, reproducible and strong manner presents challenges specific to the device design.

Many features can be incorporated into its sequential lateral flow device to ensure liquid confinement and controlled release, according to some practices. For example, the housing unit of a device of the present disclosure may be involved in the handling of liquids within the device during use. In some practices, an approximate and variable amount of liquid is included and later released from the reservoir and / or confinement area to facilitate sequential delivery of the liquid (eg, sample and reagent buffer). It must be. In some practices, the user actually adds the sample through its first entrance (eg, sample well). In some practices, the sample wets the sample receiving pad and then the transfer pad containing the binder (eg, antibody).

In some aspects, the sample receiving pad and its transfer pad do not have the capacity to completely contain the liquid sample. However, the liquid sample must still be controlled and / or retained to prevent overflow from the flow path on the capillary bed. This is because the overflow results in the passage of the sample liquid instead of passing through the pores of the nitrocellulose pad of the capillary flow bed containing the capture agent (eg, capture antibody). In addition, if the liquid sample and / or buffer is not properly contained, the sample and buffer can leave the desired flow path and pass through the interior and / or cavity of the housing unit. In either situation, the detection is compromised. Retaining and releasing the liquid in its device to independently assist in the sequential delivery of each liquid, as well as the flow of any liquid into its flow path (eg, nitrocellulose flow path) at the desired time point. Controlling is a challenge. The same challenge applies to buffer and buffer receiving pads, as well as conjugate pads.

In some practices, the housing unit may interact with the pad and facilitate confinement and release of the liquid sample and buffer into the capillary flow bed (the nitrocellulose pad) in a sequential fashion. FIG. 10 shows a schematic representation of the inner surface of a housing unit (eg, upper portion) with a series of ribs, according to some practices. In some such practices, a series of ribs are incorporated into the housing unit (eg, on the inner surface of the upper portion of the housing unit) to facilitate the sequential flow of liquid sample and buffer. .. In some practices, the ribs are approximately perpendicular and / or approximately parallel to the capillary flow bed (eg, nitrocellulose flow path).

FIG. 10 shows that the ribs can be placed on the inner surface of the upper portion of the housing unit (eg, on the sample receiving pad and / or on the buffer receiving pad) according to some practices. Is shown.

In some practices, the housing unit may include a plastic material. This is because the surface energy and / or wettability of the plastic material can contribute to its liquid confinement and / or release. For example, plastic materials may not be as capable of retaining liquids as the highly absorbent materials of capillary flow beds. Thus, plastic materials are particularly suitable for housing units due to the need to facilitate the easy release of the liquid to the capillary flow bed (eg, nitrocellulose) to the point of capillary saturation. possible. As the liquid moves through the capillary flow bed to the reservoir pad (eg, end core), the fluid contained in the housing unit passes through the sample receiving pad to the capillary flow bed and then to the capillary flow bed. It is sequentially released so as to flow toward the reservoir pad (for example, the end core material). In some practices, the ribs may retain the liquid in place until the capillary flow bed (eg, nitrocellulose) gradually becomes less saturated. In this regard, more liquid is then transferred to its transfer pad from the sample receiving pad, which remains saturated with the liquid retained by the wet housing features. The size and spacing of the ribs determines how much excess liquid can be contained in the device.

Interestingly, according to some practices, once the liquid sample and / or its buffer is depleted to the point where it is no longer saturated, the entrance to the device (eg, the sample and / or buffer). Excess liquid from the well) becomes available to its capillary flow bed. In some such practices, this process gradually desaturates the highly absorbent material of the capillary flow bed (eg, nitrocellulose pad), thus becoming a source of capillarity and liquid saturation. Continue by pulling the liquid from the combined pad. The oversaturated buffer receiving pad then saturates the conjugate pad, as described similarly for the sample receiving pad and the transfer pad. The buffer receiving pad is held in place by ribs on the inner surface of the upper portion of the housing unit just below its second inlet (ie, buffer well), according to some practices. By pulling the liquid, it is maintained in an oversaturated state.

In some practices, the liquid sample and buffer exceed the capacity of one of its pads to adequately absorb the required volume in order to perform the assay on the disclosed device. And then added to the device. In some practices, excess liquid is introduced into the device. Because the pad must "give up" the liquid to the nitrocellulose of the capillary flow bed. This oversaturation inevitably results in a tendency for the liquid to flow out within the device and / or not be sufficiently contained to prevent method failure. To prevent potential overflow in the housing unit, many structural features can be incorporated into the device (eg, in the housing unit) according to some practices.

The capillary flow bed and its reservoir pad include a major liquid flow path path in the device. Liquid samples and reagent buffers originating at the first and second inlets, respectively, are supplied to this main flow path. Once these liquids are introduced into the device, they saturate the sample receiving pad and the buffer receiving pad, respectively, and begin to pool on any wet surface. This situation presents a kinetics in which some of the liquid flows over the surface of the capillary flow bed and has no contact with the capture zone within the pores of the capillary flow bed (eg, nitrocellulose pores). Occurs.

FIG. 11 shows that, according to some practices, the capillary flow bed can be curved so that liquid enters the pore structure of the capillary flow bed (eg, nitrocellulose pores) to prevent overflow. Show that. Basically, the curvature in the capillary flow bed allows the liquid to enter the structure of the nitrocellulose pores of the capillary flow bed through capillarity.

FIG. 11 also shows that, according to some practices, the capillary flow bed is curved to prevent the liquid from flowing over the surface of the bed. In certain such practices, the capillary flowbed is curved such that the contact point of the transfer pad is about 0.02 mm to about 1 mm lower than one or more adjacent flowbed supports. Will be done. This (approximately) results in a curvature angle of about 0.05 ° to about 10 °. Its curvature promotes capillary flow and reduces flow beyond the surface of the capillary bed, resulting in excess reagent added to the wells and any liquid on the capillary flow bed pooled adjacent to the liquid reservoir. And thereby reducing local overflows that reduce the sequential delivery of fluid from its reservoir.

12a-12d show wide pinch points built into the underside of the housing unit according to some practices. In some such practices, a wide contact area is incorporated into the housing unit (on the upper portion of the housing unit) to exert pressure on the bond pad, thereby providing the reconstructed conjugate. Direct it to flow into the nitrocellulose pores of the capillary bed. It has been determined that this wide area contact can act as a wide pinch point to facilitate a more dense and discontinuous flow of conjugates through the device.

Theoretically, every point where the capillary flow bed comes into contact with the housing unit can be a position that can create capillarity. This occurs because the liquid can wet the surface of the plastic material, and once wet, its contact point becomes a point of internal overflow. In some practices, the features of the housing unit in contact with its capillary flow bed can be incorporated to prevent capillarity from creating unwanted and / or otherwise uncontrolled pathways that the liquid follows.

FIG. 4 shows an open view of a sequential lateral flow device with a capillary flow bed fixed to the lower portion of the housing unit according to some practices. In some embodiments, the housing unit (eg, the lower portion of the housing unit) may also incorporate features that represent a restricted contact portion of the capillary flow bed that is separated from the rest of the capillary flow bed. .. In certain such practices, the lower portion of the housing unit may include separate and discontinuous support blocks, as shown in FIG. In some such practices, the support block is placed under the capillary flow bed at a position corresponding to the sample receiving pad. In another embodiment, the support block is aligned under its capillary flow bed at a position corresponding to its buffer receiving pad, eg, at its second entrance. In some practices, the support block is placed under the capillary flow bed in a position corresponding to the reservoir pad.

In some practices, the support block below the first entrance is only long enough to provide a support for the capillary pad, the first entrance, and the associated pinch point. In some practices, the support block below the second entrance is only long enough to provide a support for the capillary pad, the second entrance, and the associated pinch point.

FIG. 13 shows the position of the pinch point in the housing unit with reference to the capillary flow bed according to some practices. In certain such practices, the housing unit may contain one or more pinch points in the housing unit (eg, on the inner surface of the housing unit). The purpose of the pinch point is to provide a reliable and reproducible flow path for the various components on the capillary flow bed (eg, sample receiving pad, buffer receiving pad, reservoir, conjugate pad, and transfer pad). It is to ensure that it is in contact with adjacent components so that it is established within the device. In some practices, the pinch point presses the buffer receiving pad onto the conjugate pad, thereby creating a flow path through which the buffer flows to the conjugate pad. In some practices, the pinch point presses the conjugate pad against the capillary flow bed (nitrocellulose), thereby creating a flow path through which the sample and its analyte-binder flows into the capillary flow bed. .. In some practices, the pinch point presses the sample receiving pad against the transfer pad, creating a flow path through which the sample flows from the sample receiving pad to the transfer pad. In some practices, the pinch point presses the transfer pad against the capillary flow bed, creating a flow path through which the sample flows from the transfer pad to the capillary flow bed. In some practices, the pinch point exerts sufficient pressure to facilitate a reproducible liquid flow path in a compatible material (eg, capillary flow bed). For example, the pinch point may exert sufficient force to ensure a continuous flow path, according to some practices, but does not limit and / or damage the individual components of the device.

Sequential lateral flow devices suitable for use in the methods of the present disclosure may be selected from any of the sequential lateral flow devices and / or kit implementations described above and / or below.

Reagents suitable for use in the methods, devices, and / or kits of the present disclosure include, but are not limited to:

In some practices, the methods and devices of the present disclosure include signal generators. The signal generator reports the presence of a detectable signal (eg, a visually detectable signal, a chemically detectable signal, an electromagnetically detectable signal, and / or an analyte in a test sample). It can generate a signal) that can be detected by the instrument. Various signal generators suitable for use in the methods and / or devices of the present disclosure include agents that generate signals through chemical and / or physical means. For example, the signal generator can be particles (eg, pigmented particles, latex particles, metal particles (eg, gold, silver, platinum nanoparticles, and / or colloidal metal particles), fluorescent particles, magnetic particles, chemical luminescence. Can be particles, non-metallic colloidal particles, and / or luminescent particles). Other suitable signal generators include liposomes, plastic particles and / or polymer particles, stained microorganisms, cells, enzyme substrates and / or enzymes (eg, catalytic enzymes), specific binding agents, and / or visible. Examples include, but are not limited to, arbitrary vesicles containing substances.

In some practices, when the signal-producing agent is a particle, its particle size is about 30 nm to about 120 nm in diameter, or about 40 nm to about 100 nm in diameter, or about 50 nm to about 90 nm in diameter, or about 60 nm to about 80 nm in diameter. Can be in the range of. In some practices, its particle size is about 40 nm, about 60 nm, or about 80 nm in diameter. In some practices, its particle size is about 40 nm in diameter.

In some practices, the signal generator provides a detectable signal (eg, a signal in which the binder-bound analysis is detectable in the capture zone of the device captured by the capture agent).

In some practices, the signal generating agent (eg, signal generating particles) is dried in a conjugate pad.

In some practices, the signal-generating agent is attached to and / or labeled with the first or second member of the conjugate pair. In certain such practices, the signal-generating agent is associated with and / or labeled with the first or second member of the conjugate pair, and the binder is of the conjugate pair. Attached to and / or labeled with these to the second or first member.

In some practices, the signal-generating agent is gold nanoparticles labeled with the first member of the conjugate pair (eg, avidin, streptavidin, and / or other biotin-binding proteins and / or antibodies). For example, it contains gold nanoparticles of about 40 nm, about 60 nm, or about 80 nm), while the binder is attached to a second member of its conjugate pair (eg, biotin and / or any avidin binding moiety). Can be labeled.

In some practices, the methods and devices of the present disclosure may include multiple binders. In certain such practices, each of the multiple binders is labeled with the first or second member of the conjugate pair (eg, biotin and / or any avidin binding moiety and / or molecule). obtain. In some practices, the binder may be present on the transfer pad (eg, in dry form on the transfer pad). In some practices, the binder may be included in the sample receiving pad.

In some practices, the multiple binders of the present disclosure are antibodies and / or functional fragments thereof that specifically bind to at least a partial set of common antigens among multiple Gram-negative bacteria. Antibodies that specifically bind to at least a partial set of common antigens among Gram-positive bacteria). In some practices, the binder is a polyclonal antibody (eg, one or more types of multivalent polyclonal antibodies). In other practices, the binder is a monoclonal antibody. In some practices, the plurality of binders comprises one or more types of antibodies or two or more types of antibodies, wherein each antibody is a liquid. It specifically binds to the common antigen of a subset of the bacteria in the sample.

In some practices, the polyclonal antibody binds to lipoteichoic acid (LTA). In some practices, the polyclonal antibody binds to the bacterial lipopolysaccharide structure (LPS). In some practices, at least one type of the binder specifically binds to a Gram-positive bacterial antigen, and at least one type of the binder to a Gram-negative bacterial antigen. It binds specifically. In some practices, the plurality of binders are of one or more (eg, two or more, three or more, four or more) bacteria. Can bind to a genus.

In some practices, the antibody is selected from polyclonal antibodies, monoclonal antibodies and combinations of polyclonal antibodies and monoclonal antibodies. In some practices, the antibody is polyclonal and binds to a common antigen of multiple bacteria. In some practices, the antibody is polyclonal and binds to multiple Gram-positive and / or multiple Gram-negative bacteria, and / or both common antigens. In some practices, at least one antibody is a monoclonal antibody and at least one antibody is a polyclonal antibody. In some practices, at least one antibody specifically binds to the common antigen of Gram-positive bacteria, and at least one antibody specifically binds to the common antigen of Gram-negative bacteria. In some practices, the antibody binds to one or more (eg, two or more, three or more, four or more) bacterial genera. Can be.

In some practices, such binding agents bind to antigen-containing epitopes of lipoteichoic acid (LPS) and / or lipoteichoic acid (LTA) structures of Gram-positive bacteria under physiological conditions. Contains antibodies.

Antibodies useful in the methods and devices of the present disclosure include monoclonal antibodies, polyclonal antibodies, single-stranded antibodies, synthetic antibodies, recombinant antibodies, chimeric antibodies, and / or any of the antigen-binding fragments described above (F (ab), Examples include, but are not limited to, F (ab'), F (ab') ₂ , scFv fragments and recombinant fragments). The antibody can be of non-species (eg, chicken antibody) and / or of a mammalian species, such as rabbits, rodents (mouse, rat, And guinea pigs), goats, pigs, sheep, camels and humans, but are not limited to these. The antibody can also be a humanized and / or chimeric antibody.

One of skill in the art can make any such antibody derivative using standard arts recognized in the art. For example, Jones et al. (Nature 321: 522-525 (1986)) discloses that the CDRs of human antibodies are replaced with those derived from mouse antibodies. Marx (Science 229: 455-456 (1985)) considers a chimeric antibody having a mouse variable region and a human constant region. Rodwell (Nature 342: 99-100 (1989)) considers lower molecular weight recognition elements obtained from antibody CDR information. Clackson (Br. J. Rheumator. 3052: 36-39 (1991)) includes genetically engineered monoclonal antibodies (Fv fragment derivatives, single-stranded antibodies, fusion proteins, chimeric antibodies and humanized rodent antibodies). To be considered). Reichman et al. (Nature 332: 323-327 (1988)) discloses a human antibody in which a rat hypervariable region has been grafted. Verhoeyen et al. (Science 239: 1534-1536 (1988)) teaches the grafting of mouse antigen binding sites onto human antibodies.

Preferably, the antibodies of the present disclosure are polyclonal antibodies and / or monoclonal antibodies. The production of monoclonal and polyclonal antibodies is well within the knowledge of those skilled in the art of biology (eg, Ausube et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. , 1994). Many procedures are useful in producing antibodies against the desired specific target antigen. Traditional immunotechniques and harvesting techniques result in the production of polyclonal antibodies that are directed against common determinants of the target bacterial species, including determinants such as LPS and LTA. In addition, cell hybridization techniques can be utilized to generate immortalized hybridoma cell lines that produce specific monoclonal antibodies to their target species.

Antibodies with potential utility for the widespread detection of Gram-positive bacteria include those described below: Fisher et al. , PCT Publication No. WO 98/57994; Jackson, D.C. E. et al. , Infection and Immunity 43: 800 (1984); Hamada, S. et al. et al, Microbiol. Immunol. 28: 1009 (1984); Asjord, P. et al. et al. , Acta Path. Microbiol. Immunol. Scand. Sect. C, 93: 245 (1985); McDaniel, L. et al. S. et al. , Microbial Pathogenesis 3: 249 (1987); Tadler, M. et al. B. et al. , Journal of Clinical Laboratory Analysis 3: 21 (1989); and Stuertz, K et al. , Journal of Clinical Microbiology 36: 2346 (1998).

Antibodies with potential utility for the widespread detection of Gram-negative bacteria include those described below: Nelles, M. et al. J. et al, Infect. Immun. 46: 677 (1984); Teng, N. et al. N. H. et al, Proc. Natl. Acad. Sci. USA 82: 1790 (1985); Dunn, D.M. L. et al. , Surgery 98: 283 (1985); De John-Leuvenink, J. Mol. et al, Euro. J. Clin. Microbiol. 5: 148 (1986); Bogard, W. et al. C. et al. , Infect. Immun. 55: 899 (1987); Pollack, M. et al. et al. , Bacterial Endotoxins: Pathophysiological Effects, Clinical Signal, and Physical Control. pp. 327-338 Alan R. Squirrel, Inc. (1988); Priest, B. et al. P. et al. , Surgery 106: 147 (1989); Tyler, J. et al. W. et al. , Journal of Immunological Methods 129: 221 (1990); Siegel, S. et al. A. et al. , Infect. Immun. 61: 512 (1993); Shelburne, C.I. E. et al. , J. Periodont. Res. 28: 1 (1993); Di Paradova, F. et al. E. et al. , Infect. Immun. 61: 3863 (1993); and De Kievit, T. et al. R. and Lam, J. et al. S. J. Bacteriol. 176: 7129 (1994).

The antibody used can be selected using classical techniques. Antibody specificity, degree of binding and kinetics can be characterized by empirical testing of each antibody in an empirical manner. Microtiter screening formats are well documented in the literature to aid in characterizing specific antibody responses in any given immunoassay format. Similarly, the activity of a detectably labeled antibody can be characterized by performing various chemical conjugation techniques and screening the resulting products for their optimal performance parameters. The captured antibody and detectably labeled antibody are screened against clinical isolates of bacteria from retained platelet or erythrocyte samples to match final assay performance as close as possible to the final product embodiment. Can be done. This experimental method results in the selection and optimization of antibody reagents for application in the various assay formats described below.

Monoclonal antibodies having specificity for cross-genus target on the surface of bacterial cells can be utilized in the methods of the present disclosure. In some practices, blends of monoclonal antibodies may be utilized. Polyclonal antibodies, including polyclonal antisera and / or monoclonal antibodies having broad specificity across different Gram-negative and Gram-positive species and / or polyclonal mixtures made by blending polyclonal antibodies) are described in the methods of the present disclosure and Useful in devices.

In some practices, the antibodies disclosed herein can be utilized as described and / or modified to some extent necessary to produce a useful agent.

In some practices, the methods and devices of the present disclosure may include multiple scavengers. The plurality of scavengers may be immobilized on a solid support (eg, immobilized on a capillary flow bed by covalent bond via a chemical bond), and / or the plurality of scavengers may be several. Depending on the practice, it can be immobilized via absorption.

In some practices, the scavenger is an antibody and / or a functional fragment thereof (eg, any of the antibodies described in the present disclosure). In some practices, the scavenger comprises the same type of antibody as the plurality of binders. In other practices, the scavenger comprises different types of antibodies from the plurality of binders. In some practices, the plurality of capture antibodies are in a taxon (eg, the solid support) at one or more positions on the solid support (eg, capillary flow bed). It is immobilized at one or more positions above (eg, one or more positions within the capture zone of the capillary flow bed). In some practices, the capture agent is the capture of the capillary flow bed. Immobilized at one or more positions within the zone. In some practices, the plurality of capture agents comprises one or more capture agent taxa on its solid support. The taxa are spatially separated from each other, and each taxon contains antibodies that specifically bind to at least a partial set of different common antigens in the target analyte.

In one aspect, the disclosure provides a kit for detecting and / or screening multiple targeted analytes (eg, bacteria, viruses, and / or fungi) in a liquid sample. The kit includes any one of the devices of the present disclosure, a dropper with a fixed volume calibration for the capacity of the sample receiving pad, a buffer solution and / or a buffered salt for preparing the buffer solution, and the buffer thereof. It may contain one or more reagents for pretreatment of liquid samples. The kit further includes instructions for providing procedural steps for using the device and / or control reagents as positive and / or negative controls. In some practices, the kit can be used to perform any method of the present disclosure. In some practices, the steps of detecting and / or screening multiple target analytes include the step of detecting the presence of multiple target analytes (eg, a clinically relevant amount of target analyte). To do.

Variations and modifications will be recalled to those skilled in the art after reviewing this disclosure. The disclosed features can be implemented in any combination and partial combination (including a large number of combinations and partial combinations) with one or more other features described herein. The various features described and / or illustrated above may be combined and / or incorporated into other systems, including components of any of these. In addition, certain features may be omitted and / or may not be implemented.

It is important to note that the features and / or construction and arrangement of their components as shown in the various exemplary practices are merely exemplary. Although very few practices have been described in detail in this disclosure, those skilled in the art considering this disclosure can make many modifications without essentially deviating from the teachings and advantages of the disclosed subject matter ( For example, variations in size, dimensions, structure, shape and harmony of various elements, parameter values, mounting arrangements, material use, colors, orientations, etc.) are easily recognized. For example, an element shown to be integrally formed can be constructed from a large number of parts and / or elements, the positions of the elements may be reversed and / or otherwise varied. , The nature and / or number of distinct elements and / or positions may vary and / or vary. The order and / or order of any process and / or method steps may vary according to alternative practices and / or may be reordered. Other substitutions, modifications, modifications and omissions may also be made in the design, operating conditions and arrangement of its various exemplary practices without departing from the scope of the present disclosure.

Although various practices have been described and exemplified herein, those skilled in the art will perform and / or obtain one or more of the results and / or the benefits described herein. Various other mechanisms and / or structures are readily envisioned, and each such variation and / or modification is considered to be within the scope of practice described herein. More generally, one of ordinary skill in the art will imply that any parameters, dimensions, materials, and configurations described herein are exemplary unless otherwise noted, and the actual parameters, dimensions, materials, and / Or easily recognize that the configuration depends on the particular application in which the teaching is used. One of ordinary skill in the art may recognize and / or confirm many equivalents to the specific practices described herein using simply conventional design of experiments. Accordingly, the above-mentioned practices are shown by way of example only and within the scope of the appended claims and their equivalents, the practices being specifically stated and performed in a manner different from that stated in the claims. What can be done should be understood. The implementation of this disclosure relates to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods may have such features, systems, articles, materials, kits, and / or methods with each other. If not inconsistent, it is included within the scope of the invention of the present disclosure.

In the present disclosure, the sample can be any liquid sample suspected of containing bacteria, viruses, and / or fungi. In some practices, the sample is a biological fluid (including urine, sputum, spinal fluid, ascites, blood and blood products), such as blood and / or blood products. In some practices, the blood and / or blood product is selected from the group consisting of whole blood, leukocytes, hematopoietic stem cells, platelets, erythrocytes, plasma, bone marrow and serum.

In some practices, the disclosure provides devices, kits, and methods for detecting a wide range of bacterial genera, species, and / or strains in platelet storage samples. Platelets and / or platelets are blood components responsible for coagulation and wound healing. Thrombosis has low platelet counts (eg, leukemia patients, cancer patients undergoing chemotherapy and / or radiotherapy, patients with autoimmune disease, intraoperative and / or trauma and / or infection. Infused to replenish the patient). In general, it is difficult to store and / or store platelets for long-term use. This is because platelets have a short shelf life of 5 days and must be stored at room temperature (cooled platelets are rapidly removed by the liver). Thereby, platelets are sensitive to bacterial contamination during storage, which is derived from the donor skin flora. Infusion of contaminated platelet units can cause sepsis in patients, resulting in morbidity and / or death; therefore, it is crucial to detect bacterial contamination during and before infusion of platelets.

Current practice in determining the safety of platelets for infusion relies on the use of sample storage bags 24 hours after collection and culture growth methods for bacterial detection. The culture-based test cannot rule out false negatives due to inadequate sampling of very low CFU in the early log phase of bacterial growth. Moreover, if used within hours of infusion, the culture-based test may still require a minimum number of hours for results.

In some practices, the blood and / or blood product (eg, platelets) is of donor origin for infusion to the recipient. In some practices, the sample is a dialysis sample (eg, a dialysis sample selected from hemodialysis fluids and peritoneal dialysis fluids). In some practices, the sample is a fluid in which tissue is stored, such as donor-derived tissue for transplantation into a recipient. In certain such practices, the tissue is selected from the group consisting of blood cell cultures, stem cell cultures, skin and bone and cartilage graft materials. In some practices, the sample is derived from lung, bronchoalveolar, abdominal cavity and / or arthroscopic lavage fluid. In other practices, the sample is an environmental sample (eg, water and soil). In some practices, the sample is food and / or beverage. Those skilled in the art will appreciate that if the sample source is in solid form (eg, soil and / or solid food), the sample was in contact with the liquid extract in solid form and / or in solid form. Recognize that it can be. In some practices, the sample is a biological sample (eg, urine, tears, sputum and / or cerebrospinal fluid).

In some practices, the samples of the present disclosure are pretreated via chemical and / or mechanical means. For example, in some practices, a liquid sample of appropriate volume is mixed with a pretreatment reagent such that a fragment of the cell wall structure becomes the sample. This treatment results in exposure of the binding site of the target analyte (eg, exposure of bacterial antigens and / or antigenic components). Suitable pretreatment reagents can be chemicals (eg, surfactants, chelating agents, and / or enzymes) that degrade large molecular structures and expose natural antigenic structures. The process also uses mechanical fragmentation (eg, sonication) and / or kinetic energy (eg, boiling) by mechanical means to break the cell wall into its components, thereby breaking it down. , The binding site of the binder can be exposed on the antigenic component. In other practices, the sample is base-shocked and subsequently neutralized by a neutralizing agent. For example, the sample is mixed with a base solution (eg, NaOH solution) that acts as a lysing solution to disrupt the cell wall, followed by neutralization with a neutralizing agent. Such sample pretreatment also acts to dissociate bacteria and bacterial fragments that may bind to endogenous antibodies and binding factors from the donor.

In some practices, the pretreated sample is mixed with multiple binders. In certain such practices, the plurality of binders include antibodies, such as detection antibodies (eg, polyclonal antibodies or multivalent polyclonal antibodies). In some such practices, the binder is in solution containing a neutralizing agent.

In some practices, the sample is processed before or at the same time as the contact of the sample with the antibody. In some practices, the antibody may bind to at least a partial set of common antigens in the target analyte. For example, the antibody can bind to bacterial lipoteichoic acid (LTA) or bacterial lipopolysaccharide structure (LPS). In some practices, the antibody can be an antibody that can bind to more than one genus of bacteria, viruses, and / or fungi. For example, the treatment exposes a binding site on a Gram-negative bacterial antigen and / or a Gram-positive bacterial antigen for the antibody. A binding site on a bacterial antigen, for example, cleaves the antigen from the bacterial cell wall and / or cell pad, thereby exposing the binding site; inducing the bacterium to secrete the antigen, thereby By lysing the bacterium and thereby releasing the intracellular bacterial antigen and thus exposing the binding site on the antigen; and / or conformation on the bacterial antigen. It can be exposed by inducing change and thereby exposing its binding site. Such treatments include, but are not limited to, sonication, boiling, and / or homogenization by physical means, eg, a machine of bacterial cells in the sample, using a Dawn homogenizer. Including physical destruction. The treatment also includes compounds and / or compositions (eg, surfactants), basic solutions (for alkali dissolution), acidic solutions (for acid dissolution), EDTA, EGTA, metal ions, anions, cations. , A surfactant, a chelating agent, and / or an enzyme (eg, lysostaphin, lysozyme, mutanollysin, labiase, achromopeptidase, trypsin, proteinase K, or self-solubilizer, encoded by a bacterial compound It can be the processing of the sample by chemical means (with lysogenic enzymes, and combinations thereof). The treatment exposes the binding site for the antibody on Gram-negative bacterial antigens and / or Gram-positive bacterial antigens. The treatment also dissociates the bacteria or bacterial antigens that bind to endogenous antibodies and binding factors.

It is understood that the various practices discussed herein can be combined and, in fact, be combined with any particular practice of the system, device, or method without departing from the scope of the invention. To.

1. 1. Various bacteria in a blood sample are detected using a sequential lateral flow device compared to detection with a non-sequential lateral flow device (direct assay).

2. Use of Sequential Lateral Flow Devices of the Disclosure for Bacterial Detection The purpose of this experiment is to detect bacterial antigens by performing assays in sequential and non-sequential delivery of samples and reagents via test strips. It was to compare abilities. The sample was prepared from a 10-fold dilution of Pseudomonas aeruginosa ATCC 27853. Detection of bacterial antigens was facilitated by scanning signals generated from colloidal gold particles immobilized on nitrocellulose via an immunological assay within a striped band of immobilized capture antibody. Quantification of immobilized colloidal gold is available in LFStudio software ver. It was facilitated by scanning with a Qiagen ESEQuant LR3 reader using 3.6.0 and a baseline set to 30 units. The quantification of Pseudomonas aeruginosa ATCC 27853 bacteria was performed using the dilution plate counting (DPC) method. This is a representative method used by microbiologists and is known to those of skill in the art.

In this dilution plate counting method, 100 μL platelet samples containing Pa 27853 were serially diluted 10-fold with fresh platelet samples. After a 36 hour incubation time, the colony forming units (CFU) were counted to determine the number of bacteria contained in the first sample. These 10-fold diluted samples were then run in an assay utilizing either sequential or non-sequential delivery of samples and reagents to the test strip. All components, including the prepared sample, were identical for direct comparison between sequential and non-sequential practices. The results of this experiment are shown in FIG.

Variations and modifications will be recalled to those skilled in the art after reviewing this disclosure. The disclosed features may be implemented in any combination and partial combination (including multiple dependent and partial combinations) with one or more of the other features described herein. .. The various features described and / or illustrated above may be combined and / or incorporated in other systems, including any of their components. Examples of changes, substitutions, and quality changes are visible to those of skill in the art and can be made without departing from the scope of the information disclosed herein. In addition, certain features may be omitted and / or may not be implemented. All references cited are incorporated herein by reference in their entirety and may be incorporated into this application.

Claims (52)

A method for detecting multiple target analytes in a liquid sample using a lateral flow device.
(A) The liquid sample is contacted with a plurality of capture agents and a plurality of binders placed on a solid support in the lateral flow device under conditions that allow the formation of at least one sandwich complex. In the step of allowing, the at least one sandwich complex is one or more of the plurality of binders, one or more of the target analytes, and of the scavengers. Containing one or more, where each of the plurality of binders is tagged with one member of a conjugate pair, the plurality of binders and the plurality of scavengers comprises a polyclonal antibody. The plurality of binders and / or the plurality of capture agents may bind to at least one antigen common to at least a subset of the target analyte, step;
(B) To form the detection complex, the sandwich complex and the non-enzymatic signal generator are brought into contact under conditions that allow the signal generator to bind to the binder of the sandwich complex. In the step, the signal-generating agent is here tagged with a second member of the conjugate pair, and binding of the signal-generating agent to the binder of the sandwich complex is said in the liquid sample. It exhibits the presence of one or more of the target analytes, and the lateral flow device inhibits the signal-producing agent from contacting the plurality of binders prior to the formation of the sandwich complex. The step, which is adapted as described above; and (c) detecting the signal generated by the formation of the detection complex to determine the presence of the plurality of target analytes.
A method of including. The contacting step in the step (a) is
The liquid sample and the plurality of binders are contacted under conditions that allow the formation of a first complex between at least some of the target analytes and at least some of the binders. And to form the sandwich complex, the first complex and the plurality of scavengers are applied only after the liquid sample has formed the first complex. To make contact with,
The method according to claim 1, comprising the above. The contacting step in the step (a) is
The liquid sample and the plurality of capture agents are brought into contact with each other under conditions that allow the formation of a complex between at least some of the target analytes and at least some of the capture agents; And to form the sandwich complex, contacting the complex with the plurality of binders such that the binder is in contact with the target analyte only after the formation of the complex.
The method according to claim 1, comprising the above. The lateral flow device is placed between a sample receiving pad in the sample receiving zone for introducing the liquid sample into the lateral flow device and a capillary flow bed in the solid support that facilitates the flow of the liquid sample. The method of any one of claims 1-3, which comprises a substantially impermeable backing, thereby reducing the backflow of the liquid sample in the proximal direction of the lateral flow device. The plurality of scavengers comprises one or more types of scavengers such that each specifically binds to at least the common antigen of the partial set of target analytes in the liquid sample. The method according to any one of claims 1 to 4, which is adapted to. The method according to any one of claims 1 to 5, wherein the plurality of scavengers bind to a Gram-positive bacterial antigen and / or a Gram-negative bacterial antigen. The plurality of scavengers comprises one or more scavenger taxa on the solid support, the taxa being spatially separated from each other, and each taxon being a different target analyte. The method according to any one of claims 1 to 6, comprising an antibody that binds to. The plurality of binders comprises one or more types of binders such that each specifically binds to at least the common antigen of the partial set of the target analyte in the liquid sample. The method according to any one of claims 1 to 7, which is adapted to. The method according to any one of claims 1 to 8, wherein the plurality of binders bind to a Gram-positive bacterial antigen and / or a Gram-negative bacterial antigen. The method of any one of claims 1-9, wherein at least some of the binders are antibodies of the same type as at least some of the scavengers. The method of any one of claims 1-10, wherein at least some of the binders are antibodies of a different type than at least some of the scavengers. The lateral flow device comprises a housing unit, wherein the upper inner surface of the housing unit includes a series of ribs to include liquid beyond the capacity of the sample receiving pad, thereby the excess. The method according to any one of claims 1 to 11, wherein the fluid is prevented from overflowing from the sample receiving pad. Each of the plurality of binders is labeled with the first member of the conjugate pair, and the signal generator is labeled with the second member of the conjugate pair, any one of claims 1-2. The method described in the section. 13. The method of claim 13, wherein the conjugate pair comprises biotin and a biotin-binding protein, and each of the plurality of binders or each of the plurality of signal generators is labeled with biotin. The method of claim 14, wherein the biotin-binding protein is selected from the group consisting of avidin, neutravidin, anti-biotin antibody, streptavidin, and other biotin-binding proteins. The method according to any one of claims 1 to 15, wherein the signal generator is selected from the group consisting of metal particles, fluorescent dyes, and latex particles. The method of any one of claims 1-15, wherein the sample is pretreated using base degradation followed by neutralization. 17. The method of claim 17, wherein the pretreated sample is mixed with a plurality of binders, if necessary. The method of any one of claims 1-18, wherein the plurality of targeted analytes comprises a bacterial antigen, a viral antigen, and / or a fungal antigen. A lateral flow device for detecting multiple target analytes in a liquid sample.
A housing unit that includes an inner surface that defines a cavity within the housing unit;
A capillary flow bed in the cavity, wherein the capillary flow bed is configured to transport the sample from the proximal region of the capillary flow bed to the distal region of the capillary flow bed. Capillary flow bed;
Buffer receiving pad A (P _B) and buffer receiving zone comprising a conjugate pad (P _C), the conjugate pad comprises a signal producing agent to provide a detectable signal, buffers receptor zone;
Capture zone (C) containing multiple immobilized capture agents;
A sample receiving zone located between the buffer receiving zone and the capture zone, the sample receiving zone, the sample receiving pad (P _S), transfer pad (P _T), and with said sample receiving zone The transfer pad comprises a substantially opaque backing that is located between the capillary flow bed and extends at least partially under the sample receiving pad and the transfer pad, the transfer pad being specific to multiple target analytes. Sample receiving zone containing multiple binders that bind specifically;
Reservoir pad (R) located in the distal region of the capillary flow bed;
The first entrance (I ₁ ) in the housing for introducing the liquid sample into the sample receiving pad, where the impermeable backing is the liquid sample and the liquid sample in the sample receiving zone. A first entrance that blocks contact with the capillary flow bed, thereby reducing backflow of the sample in the proximal direction;
A second inlet (I ₂ ) in the housing for introducing the buffer into the buffer receiving pad, where the buffer mobilizes the signal generator and mobilizes the signal generator. A second entrance; as well as across the capture zone for observing the detectable signal produced by the interaction of the binder with the signal generator in the presence of the target analyte. Reading window specified in the housing unit;
Including
Here, the reservoir pad is adapted to pull the liquid sample and the mobilized signal generator in the distal flow direction, and the device is said to have the mobilized signal generator before flowing into the capture zone. A lateral flow device configured to allow a sample to flow along the capillary flow bed into the capture zone. The lateral flow device according to claim 20, wherein the sample receiving zone, the buffer receiving zone, the capturing zone, and the reservoir pad are arranged from the proximal region to the distal region as follows:
The inner surface of the housing unit includes a series of ribs to accommodate the excess fluid, thereby preventing the excess fluid from overflowing the sample receiving pad or the buffer receiving pad. The lateral flow device according to claim 20 or 21. 22. The lateral flow device of claim 22, wherein the series of ribs is placed on the sample receiving pad on the upper inner surface of the housing unit. 23. The lateral flow device of claim 23, wherein the series of ribs is placed on the buffer receiving pad on the upper inner surface of the housing unit. The inner surface of the housing unit comprises at least one pinch point that presses the buffer receiving pad against the conjugate pad to create a flow path through which the buffer flows from the buffer receiving pad to the conjugate pad. , The lateral flow device according to any one of claims 20 to 24. 20. The inner surface of the housing unit comprises at least one pinch point that presses the sample receiving pad against the transfer pad to create a flow path through which the sample flows from the sample receiving pad to the transfer pad. The lateral flow device according to any one of 24. The inner surface of the housing unit comprises at least one pinch point that presses the transfer pad against the flow path bed to create a flow path through which the sample flows from the transfer pad to the capillary flow bed. The lateral flow device according to any one of the above. The lateral flow device according to any one of claims 20 to 27, wherein the capillary flow bed is slightly curved to promote capillarity and minimize overflow. The capture agents in the capture zone contain one or more types of capture agents, each adapted to at least a partial set of common antigens of the target analyte in the liquid sample. The lateral flow device according to any one of claims 20 to 28. The lateral flow device according to any one of claims 20 to 29, wherein the plurality of scavengers bind to a Gram-positive bacterial antigen and / or a Gram-negative bacterial antigen. The lateral flow device according to any one of claims 20 to 30, wherein the plurality of scavengers include an antibody. The lateral flow device according to any one of claims 20 to 31, wherein the plurality of scavengers include one or more of a polyclonal antibody and / or a monoclonal antibody. The plurality of scavengers comprises one or more scavenger taxa on the capillary flow bed, the taxa being spatially separated from each other, and each taxon having a different target or analysis. The lateral flow device according to any one of claims 20 to 32, which comprises an antibody that binds to an object. The lateral flow device according to any one of claims 20 to 33, wherein the plurality of binders bind to a Gram-positive bacterial antigen and / or a Gram-negative bacterial antigen. The lateral flow device according to any one of claims 20 to 34, wherein the plurality of binders include an antibody. The lateral flow device according to any one of claims 20 to 34, wherein the plurality of binders are selected from one or more of a polyclonal antibody and / or a monoclonal antibody. Each of the plurality of binders is labeled with the first member of the conjugate pair and the signal generator is labeled with the second member of the conjugate pair, any one of claims 20-36. Lateral flow device described in section. 37. The lateral flow device of claim 37, wherein the conjugate pair comprises biotin and a biotin-binding protein, and either the binder or the signal-generating agent is labeled with biotin. The lateral flow device according to claim 38, wherein the biotin-binding protein is selected from the group consisting of avidin, neutravidin, anti-biotin antibody, streptavidin, and other biotin-binding proteins. The lateral flow device according to any one of claims 20 to 39, wherein the signal generator is selected from the group consisting of metal particles, fluorescent dyes, and latex particles. The lateral flow device according to any one of claims 20 to 40, wherein the capillary flow bed contains nitrocellulose. The lateral flow device according to any one of claims 20 to 40, wherein the sample receiving pad, the buffer receiving pad, the reservoir pad, and the conjugate pad each contain a highly absorbent material. The lateral flow device according to claim 42, wherein the highly absorbent material is selected from the group consisting of porous paper, polypropylene, polyester, polyethylene, glass fiber, cellulose blends, and combinations thereof. The lateral flow device according to any one of claims 20 to 43, wherein the liquid sample is pretreated using base decomposition followed by neutralization. The lateral flow device of claim 44, wherein the pretreated liquid sample is mixed with a plurality of binders, if necessary. The lateral flow device according to any one of claims 20 to 45, wherein the plurality of target analyzes include a bacterial antigen, a viral antigen, or a fungal antigen. A method for detecting a plurality of target analytes in a liquid sample using the lateral flow device according to any one of claims 20 to 46. The detection step is
(A) A step of introducing the liquid sample into the lateral flow device through the first inlet, where the liquid sample flows from the sample receiving pad to the transfer pad and said. The liquid sample is prevented from contacting the capillary flow bed by the backing prior to contacting the distal portion of the transfer pad;
(B) By forming a first complex between at least some of the binders in the transfer pad and, if present in the sample, at least some of the target analytes. Here, the first complex is pulled through the capillary flow bed towards the capture zone by a capillary force generated by the reservoir pad located in the distal region of the capillary flow bed. ;
(C) The signal generator mobilized after introducing a buffer into the second inlet of the lateral flow device, after the sample has flowed past the capture zone and past the visual window. Mobilize the signal generator after introducing the liquid sample so that the liquid sample flows into the capture zone; and (d) read the detectable signal generated in the capture zone through the reading window.
47. The method of claim 47. The lateral flow device according to any one of claims 20 to 46, wherein the method for detecting the plurality of target analytes according to any one of claims 1 to 19 is performed. A kit for detecting one or more target analytes in a liquid sample, comprising the lateral flow device according to any one of claims 20-46. The kit of claim 50, further comprising a buffer solution or a buffer salt for preparing the buffer solution, and / or one or more reagents for pretreating the liquid sample. The kit according to claim 50 or 51, wherein the kit is used to perform the method according to any one of claims 1 to 19.